Methods of diagnosing acute cardiac disorders using bnp-sp

ABSTRACT

The invention provides methods for predicting, diagnosing or monitoring acute cardiac disorders, cardiac transplant rejection, or distinguishing acute cardiac disorders from pulmonary disorders, by measuring BNP signal peptide levels in a sample taken from a subject shortly after onset of, or presentation with the disorder or transplant rejection.

RELATED APPLICATIONS

This application is a continuation of commonly owned, co-pending U.S.patent application Ser. No. 12/381,100, filed 6 Mar. 2009, which is aU.S. national stage application based on commonly owned PCT applicationno. PCT/NZ2007/000265, filed on 7 Sep. 2007 and published on 13 Mar.2008 as WIPO publication no. WO/2008/030122, which is based on commonlyowned U.S. provisional patent application Ser. No. 60/842,649, filed on7 Sep. 2006. This application claims the benefit of and priority to eachof the foregoing patent applications for any and all purposes, and eachof them is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to BNP signal peptide (BNP-SP) and its use in theprognosis, diagnosis and monitoring of acute cardiac disorders includingacute coronary syndromes in a subject resulting in releasing ofbiomarker into the circulation. More particularly, the invention relatesto methods of predicting, diagnosing or monitoring and acute a cardiacdisorder in a subject by measuring BNP-SP levels in a sample takenshortly after onset of, or at clinical presentation with the disorder.

BACKGROUND

Acute cardiac disorders including acute coronary syndromes (ACS)encompass a wide spectrum of cardiac ischemic events ranging fromunstable angina through to acute myocardial infarction (AMI). AMIpresents as the most serious of these events and therefore requiresrapid and accurate diagnosis. Patients who present with two or more ofthe described features (a history of ischemic chest discomfort,evolutionary changes on serial electrocardiogram (ECG) traces and a riseand fall in plasma cardiac biomarkers) are clearly identified asundergoing AMI.¹ However, a significant proportion of patients (40%-50%)who present with suspected AMI do not have serial changes on ECG, ortypical symptoms thus placing heavy emphasis on circulating biomarkerconcentrations for accurate diagnosis.²⁻⁴

Accurate early diagnosis of myocardial infarction facilitates promptintroduction of reperfusion treatment, including effective percutaneousor thrombolytic revascularization and adjunctive at reducing mortalityand morbidity with each hour of delay in diagnosis and management.²⁻⁴Given the need for accelerated decision-making in this clinicalsituation, there is considerable interest in the identification ofcirculating biomarkers providing an early and specific diagnosis ofacute cardiac disorders, particularly AMI.

A number of biomarkers have been proposed for this purpose, includingcreatine kinase-MB (CK-MB), troponin T (TnT), troponin I (TnI) andmyoglobin, but there are limitations to their use. Time to detectable orabnormal elevation of plasma cardiac biomarkers can be 6 hours(myoglobin, CK-MB) to 12 hours (TnT, TnI) with peak levels not occurringuntil 24-48 hours after onset of injury, imposing a window of delay uponprecise diagnosis and treatment.¹⁻⁴ Furthermore, both myoglobin andCK-MB are non-specific and can be secreted from extra-cardiac sources;especially during trauma or surgery.¹ Other biomarkers useful for thispurpose are BNP (preproBNP 103-134) and N-BNP (preproBNP (27-134) whichis also known as NT-proBNP (see FIG. 1). Both peptides are secreted intothe circulation.

Measurement of plasma concentrations of BNP and N-BNP early post-AMI haspowerful prognostic value^(2,6,7) and incorporation of plasmaconcentrations of these peptides into treatment regimes cansignificantly improve clinical outcomes of patients with heart failure.⁸This is particularly true of N-BNP which has a half-life some 14-foldlonger than BNP⁵ and thus provides additional important informationregarding long term cardiac performance after AMI.

As with the cardiac biomarkers above, BNP and N-BNP may not reachdetectable or abnormal levels for 6 to 12 hours after onset of injury,with peak levels not occurring until 24 to 48 hours after onset. Thelong term diagnostic/predictive powers of BNP and N-BNP therefore lackthe accompanying power of a specific marker providing early specificdiagnosis of acute cardiac disorders such as acute cardiac injury withinthe first few hours of clinical presentation. A need thereof exists forsuch an early marker.

More recently, it has been suggested that BNP-SP may be useful indiagnosing heart disease (US 2005/0244904, WO 2005/052593). It isgenerally indicated that levels of BNP-SP will be higher in heartfailure patients than normal patients. No time course information as towhen to measure BNP-SP levels is provided. It is stated that BNP-SPlevels are elevated in conjunction with N-BNP.

It is an object of the present invention to go some way towardsfulfilling the need for an early marker of acute cardiac disorders,and/or to at least provide the public with a useful choice.

SUMMARY OF THE INVENTION

Human B-type natriuretic signal peptide (BNP-SP) or preproBNP (1-26) isa 26 amino acid peptide cleaved from preproBNP (1-134) SEQ ID NO: 1.BNP-SP is shown separately in SEQ ID NO:21.

The applicants have surprisingly discovered that the circulatingconcentration of BNP-SP is highest in the first few hours followingonset of, or at clinical presentation with suspected acute coronarysyndromes (ACS). Peaks are in the order of four to ten times higher,commonly five to eight times higher than normal control populations inthese first hours.

Accordingly, in a first aspect the present invention provides a methodfor predicting, diagnosing or monitoring an acute cardiac disorder (ACD)in a subject, the method comprising:

measuring the level of BNP-SP in a biological sample obtained from thesubject within two hours of onset of the ACD, or within two hours ofpresentation with the ACD; and comparing the level of said BNP-SP withthe BNP-SP level from a control wherein a measured level of BNP-SPhigher than the control level is indicative of ACD.

The invention also provides a method for monitoring a response totreatment of an acute cardiac disorder (ACD) in a subject, the methodcomprising measuring the level of BNP-SP in a biological sample obtainedfrom the subject within two hours of onset of the ACD or within twohours of presentation with the ACD; and comparing the level of saidBNP-SP with the BNP-SP level from a control, wherein a change in themeasured level of BNP-SP from the control level is indicative of aresponse to the treatment.

In another aspect, the invention also provides a method for predicting,diagnosing or monitoring a cardiac transplant rejection episode in asubject, the method comprising measuring the level of BNP-SP in abiological sample obtained from a subject within two hours of hearttransplant and comparing the level of said BNP-SP with the BNP-SP levelfrom a control, wherein a measured level of BNP-SP higher than thecontrol level is indicative of transplant rejection.

The invention also provides a method of distinguishing between apulmonary disorder and an acute cardiac disorder (ACD) in a subject, themethod comprising measuring the level of BNP-SP in a biological sampleobtained from a subject within two hours of presentation with thedisorder; and comparing the level of said BNP-SP with the BNP-SP levelfrom a control wherein a measured level of BNP-SP higher than thecontrol level is indicative of ACD.

The invention also provides a method for predicting, diagnosing ormonitoring an acute cardiac disorder (ACD), cardiac transplantrejection, or ACD/pulmonary disorder in a subject, the method comprisingmeasuring the level of BNP-SP in a biological sample obtained from thesubject within the first two hours of onset of, or clinical presentationwith ACD, cardiac transplant rejection or ACD/pulmonary disorder.

Preferably, the measured level of BNP-SP is compared with the BNP-SPlevel from a control wherein a measured level of BNP-SP higher than thecontrol level is indicative of ACD or cardiac transplant rejection.

Preferably, the methods of the invention are in vitro methods.

In one embodiment, the measurement of BNP-SP levels is carried outwithin one hour, of onset or clinical presentation, preferably within 30minutes.

Preferably, the biological sample is blood, saliva, interstitial fluid,plasma, urine, serum or heart tissue.

In one embodiment, the measuring step comprises detecting bindingbetween BNP-SP and a binding agent that selectively binds BNP-SP. Thebinding agent is preferably an antibody or antibody fragment. Mostcommonly, the antibody is a monoclonal, polyclonal or humanizedantibody. Monoclonal antibodies are preferred

In an alternate embodiment, the levels of BNP-SP are measured using massspectroscopy.

The BNP-SP which is selectively bound by the antibody is the full lengthhuman BNP-SP molecule (SEQ ID NO:21) or an antigenic variant or fragmentthereof. Preferably, the fragment is at least five amino acids inlength. Desirably, the antibody binds the N-terminus or the C-terminusof BNP-SP.

Specific antigenic peptides which the binding agent selectively bindsinclude human BNP-SP (1-10) (SEQ ID NO:13), BNP-SP (1-17) (SEQ IDNO:15), BNP-SP (3-15) (SEQ ID NO:23), BNP-SP (17-26) (SEQ ID NO:19),BNP-SP (12-23) (SEQ ID NO:17) and BNP-SP (1-26) (SEQ ID NO:21) orvariants thereof.

Binding of BNP-SP is preferably measured using antibodies or antibodyfragments that are immobilised on a solid phase.

Levels of BNP-SP may usefully be measured with an assay selected fromMA, ELISA, fluoroimmunoassay, immunofluorometric assay, massspectrometry and immunoradiometric assay.

Accordingly, the invention also provides an assay for BNP-SP in abiological sample obtained from a subject within two hours from onsetof, or within two hours of clinical presentation with ACD, cardiactransplant rejection, or ACD/pulmonary disorder, the assay comprisingdetecting and measuring the level of BNP-SP in the sample using anyknown methods.

Preferably, the assay is an in vitro assay.

The methods of the invention may further comprise measuring the level ofone or more non-BNP-SP Markers of said ACD, or cardiac transplantrejection, or ACD/pulmonary disorder and comparing the levels againstmarker levels from a control wherein a deviation in the measured levelfrom the control level of non-BNP-SP marker, together with a measuredlevel of BNP-SP which is higher than the control level of BNP-SP, ispredictive or diagnostic of the ACD, or can be used to monitor said ACD,cardiac transplant rejection or ACD/pulmonary disorder.

Markers for use in the context of acute coronary syndrome includetroponin T, troponin I, creatine kinase MB, myoglobin, BNP, NT-BNP, LDH,aspartate aminotransferase, and heart specific fatty acid bindingprotein (H-FABP).

In another aspect, the present invention also provides a BNP-SP bindingagent that selectively binds BNP-SP or an antigenic fragment or variantthereof for use in predicting, diagnosing or monitoring an acute cardiacdisorder (ACD), cardiac transplant rejection or ACD/pulmonary disorderin a subject, wherein the ACD, cardiac transplant rejection orACD/pulmonary disorder is characterised by the appearance of BNP-SP in abiological sample obtained from the subject within two hours of onsetof, or within two hours of clinical presentation with ACD, cardiactransplant rejection or ACD/pulmonary disorder.

In one embodiment, the binding agent is preferably an antibody orfragment thereof.

In another embodiment, the binding agent is any solid or non-solidmaterial capable of binding BNP-SP.

The invention is also directed to the use of BNP-SP binding agent in themanufacture of a prognostic, diagnostic or monitoring tool for assessingan acute cardiac disorder (ACD), cardiac transplant rejection orACD/pulmonary disorder in a subject, wherein assessment is carried outwithin two hours of onset of, or within two hours of clinicalpresentation with ACD, cardiac transplant rejection or ACD/pulmonarydisorder.

The invention also relates to a use of the invention wherein theprognostic, diagnostic or monitoring tool is calibrated to measureBNP-SP levels in the range of from 0.1 to 500 pmol/L, preferably 1 to400 pmol/L, preferably 10 to 350 pmol/L, preferably 20 to 300 pmol/L,preferably 25 to 250 pmol/L, preferably 30 to 180 pmol/L, preferably 35to 150 pmol/L, and preferably 40 to 120 pmol/L.

In another aspect, the invention provides a kit for predicting,diagnosing or monitoring an acute cardiac disorder (ACD), cardiactransplant rejection or ACD/pulmonary disorder comprising a BNP-SPbinding agent of the invention, wherein the kit is for use with abiological sample obtained from a subject within two hours of onset of,or clinical presentation with ACD, cardiac transplant rejection orACD/pulmonary disorder.

The invention also provides a kit for predicting, diagnosing ormonitoring an acute cardiac disorder (ACD) comprising a binding agent ofthe invention, wherein the kit is calibrated to measure BNP-SP levels inthe range of 0.1 to 500 pmol/L, preferably 1 to 400 pmol/L, preferably10 to 350 pmol/L, preferably 20 to 300 pmol/L, preferably 25 to 250pmol/L, preferably 30 to 180 pmol/L, preferably 35 to 150 pmol/L, andpreferably 40 to 120 pmol/L.

Preferably, the kit also includes instructions for predicting,diagnosing or monitoring ACD, cardiac transplant rejection, orACD/pulmonary disorder in a subject within two hours of onset, orclinical presentation, from the BNP-SP level measured in the biologicalsample obtained within two hours of onset or clinical presentation.

In another aspect, the invention relates to a nucleic acid moleculeencoding a BNP-SP of the invention wherein said nucleic acid is selectedfrom

-   -   (a) SEQ ID NO:14;    -   (b) SEQ ID NO:16;    -   (c) SEQ ID NO:18;    -   (d) SEQ ID NO:20;    -   (e) a complement of any one of (a) to (d);    -   (f) a sequence of at least 15 nucleotides in length, capable of        hybridising to the sequence of any one of (a) to (e) under        stringent conditions with the proviso that the sequence is not        ccagtgcacaagctgcttggggaggcgaga or SEQ ID NO: 22.

The invention also provides a genetic construct comprising a nucleicacid molecule of the invention, a vector comprising the geneticconstruct, a host cell comprising the genetic construct or vector, apolypeptide encoded by a nucleic acid molecule of the invention, anantibody which selectively binds a polypeptide of the invention, and amethod for recombinantly producing a polypeptide of the invention.

Accordingly, in another aspect the invention provides an isolated BNP-SPpolypeptide selected from

-   -   (a) SEQ ID NO:13;    -   (b) SEQ ID NO:15;    -   (c) SEQ ID NO:17; and    -   (d) SEQ ID NO:19.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described with reference to the figures in theaccompanying drawings in which

Figures

FIG. 1 is a schematic diagram outlining the processing of humanpreproBNP resulting in generation of free signal, N-BNP and BNPpeptides;

FIG. 2A is a single letter notation format of preproBNP sequences inseven species. The signal peptide region is in bold and underlined;

FIG. 2B is a Clustal W version 1.83 JALVIEW multiple sequence alignmentof the prepoBNP signal peptide sequences. The default Clustal Wparameters were used in this alignment as follows: DNA Gap OpenPenalty=15.0; DNA Gap Extension Penalty=6.66; DNA matrix=Identity;Protein Gap Open Penalty=10.0; Protein Gap Extension Penalty=0.2;Protein Matrix=Gonnet; Protein/DNA ENDGAP=−1; Protein/DNA GAPDIST=4. Theamino acids were submitted in the Pearson (fasta) format.⁹

FIG. 3 shows the results of a radioimmunoassay with human plasmaextracts (open squares) dilute in parallel with the BNP-SP standardcurve (filled circles);

FIG. 4 shows the results of a radioimmunoassay demonstrating that plasmaconcentrations of BNP-SP in healthy humans do not show any correlationwith age;

FIG. 5 shows the results of a radioimmunoassay demonstrating that plasmalevels of BNP and N-BNP in normal healthy humans (n=13) show closecorrelation (left panel) whereas BNP-SP concentrations do not correlatewith N-BNP (right panel);

FIG. 6 shows the results of a radioimmunoassay SEHPLC (top panel) andRPHPLC bottom panel) analysis of BNP-SP in human plasma which suggestsBNP-SP elutes close to synthetic BNP-SP (downward arrow, bottom panel);

FIG. 7. Radioimmunoassay results showing Upper panel: Concentrations ofBNP-SP (open hexagon), BNP (filled circles) and N-BNP (open squares) inplasma drawn from AME patients (n=10) at the times shown from hospitaladmission. In contrast with BNP and N-BNP levels which peaked at 24hours post-admission, highest levels of BNP-SP were seen at admission,being some 7-fold higher on average than levels measured in normalhealthy individuals (open circle). Lower panel: matched, time courseconcentration profiles of CK-MB, myoglobin and TnT in the same patientsin upper panel;

FIG. 8. Shows a table of cross reactivity data of BNP-SP antiserum; and

FIG. 9. Is a bar graph showing circulating BNP-SP concentrations inpatients are derived from a cardiac source.

DEFINITIONS

Acute Cardiac Disorder (ACD), includes but is not limited to: acutecoronary syndromes: (AMI) with ST-elevation on presenting ECG, unstableangina, and acute non ST-elevated MI; cardiac ischemia; acute cardiacinjury; acute cardiac damage resulting from acute drug toxicity, acutecardiomyopathies, and cardiac transplant rejection. Full descriptive,definitions of these disorders are found in reference 1.

ACD/pulmonary disorder refers to a subject with an undiagnosed, orsuspected ACD or pulmonary disorder.

Acute coronary syndromes (ACS) encompasses a wide spectrum of cardiacischemia events including unstable angina, acute myocardial infarct withST-elevation on presenting electrocardiogram (ECG), and acute myocardialinfarction without ST-segment elevation on ECG.

The term “antibody” refers to an immunoglobulin molecule having aspecific structure that interacts (binds) specifically with a moleculecomprising the antigen used for synthesizing the antibody or with anantigen closely related to it. An antibody binds selectively orspecifically to a BNP-SP polypeptide of the invention if the antibodybinds preferentially to the BNP-SP e.g. has less than 25%, preferablyless than 10%, preferably less than 1% cross-reactivity with anon-BNP-SP polypeptides. Usually, the antibody will have a bindingaffinity (dissociation constant (Kd) value), for the antigen of no morethan 10⁻⁷M, preferably less than about 10⁻⁸M, preferably less than about10⁻⁹M. Binding affinity may be assessed using surface plasma resonance.

Biological sample as used herein means any sample derived from a subjectto be screened. The sample may be any sample known in the art in whichthe BNP-SP can be detected. Included are any body fluids such as plasma,blood, saliva, interstitial fluid, serum, urine, synovial,cerebrospinal, lymph, seminal, amniotic, pericardial fluid and ascites,as well as tissues such as cardiac tissues but not limited thereto. Alsoincluded are samples from normal healthy subjects with no clinicalhistory of acute cardiac disorders.

The term BNP-SP refers to the complete 26 amino acid BNP signal peptidefor the human prepro BNP sequence (SEQ ID NO: 1) BNP-SP is shownseparately in SEQ ID NO:21. Also encompassed within the term BNP-SP is avariant or fragment of BNP-SP.

The term “comprising” as used in this specification and claims means“consisting at least in part of”; that is to say when interpretingstatements in this specification and claims which include “comprising”,the features prefaced by this term in each statement all need to bepresent but other features can also be present. Related terms such as“comprise” and “comprised” are to be interpreted in similar manner.

The term “polynucleotide(s),” as used herein, means a single ordouble-stranded deoxyribonucleotide or ribonucleotide polymer of anylength, and include as non-limiting examples, coding and non-codingsequences of a gene, sense and antisense sequences, exons, introns,genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes,recombinant polynucleotides, isolated and purified naturally occurringDNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acidprobes, primers, fragments, genetic constructs, vectors and modifiedpolynucleotides. Reference to a nucleic acid molecule is to be similarlyunderstood.

A “fragment” of a polynucleotide sequence provided herein is asubsequence of contiguous nucleotides that is capable of specifichybridization to a target of interest, e.g., a sequence that is at least10 nucleotides in length. The fragments of the invention comprise 10,preferably 15 nucleotides, preferably 16, preferably 17, preferably 18,preferably 19, preferably 21, preferably 22, preferably 23, preferably24, preferably 25, preferably 26, preferably 27, preferably 28,preferably 29, preferably 30, preferably 31, preferably 32, preferably33, preferably 34, preferably 35, preferably 36, preferably 37,preferably 38, preferably 39, preferably 40, preferably 41, preferably42, preferably 43, preferably 44, preferably 45, preferably 46,preferably 47, preferably 48, preferably 49, preferably 50, preferably51, preferably 52, preferably 53, preferably 54, preferably 55,preferably 56, preferably 57, preferably 58, preferably 59, preferably60, preferably 61, preferably 62, preferably 63, preferably 64,preferably 65, preferably 66, preferably 67, preferably 68, preferably69, preferably 70, preferably 71, preferably 72, preferably 73,preferably 74, preferably 75, preferably 76, preferably 77 contiguousnucleotides of a polynucleotide of SEQ ID NO:22. A fragment of apolynucleotide sequence can be used as a primer, a probe, included in amicroarray, or used in polynucleotide-based selection methods herein.

The term “primer” refers to a short polynucleotide, usually having afree 3′0H group, that is hybridized to a template and used for primingpolymerization of a polynucleotide complementary to the target.

The term “probe” refers to a short polynucleotide that is used to detecta polynucleotide sequence, that is complementary to the probe, in ahybridization-based assay. The probe may consist of a “fragment” of apolynucleotide as defined herein.

The term “polypeptide”, as used herein, encompasses amino acid chains ofany length, but preferably at least 5 amino acids, preferably at least6, preferably at least 7, preferably at least 8, preferably at least 9,preferably at least 10, preferably at least 11, 12, preferably at least13, preferably at least 4′4, preferably at least 15, preferably at least16, preferably at least 17, preferably at least 18, preferably at least19, preferably at least 20, preferably at least 21, preferably at least22, preferably at least 23, preferably at least 24, preferably at least25, and preferably all 26 amino acids of the full-length BNP-SP protein(SEQ ID NO:21), in which amino acid residues are linked by covalentpeptide bonds. Polypeptides useful in the present invention may bepurified natural products, or may be produced partially or wholly usingrecombinant or synthetic techniques. The term may refer to apolypeptide, an aggregate of a polypeptide such as a dimer or othermultimer, a fusion polypeptide, a polypeptide fragment, a polypeptidevariant, or derivative thereof.

A “fragment” of a polypeptide is a subsequence of the polypeptide thatperforms a function that is required for the biological activity orbinding and/or provides three dimensional structure of the polypeptide.The term may refer to a polypeptide, an aggregate of a polypeptide suchas a dimer or other multimer, a fusion polypeptide, a polypeptidefragment, a polypeptide variant, or derivative thereof capable ofperforming the above signal peptide activity.

The term “isolated” as applied to the polynucleotide or polypeptidesequences disclosed herein is used to refer to sequences that areremoved from their natural cellular environment. An isolated moleculemay be obtained by any method or combination of methods includingbiochemical, recombinant, and synthetic techniques. The polynucleotideor polypeptide sequences may be prepared by at least one purificationstep.

The term “recombinant” refers to a polynucleotide sequence that isremoved from sequences that surround it in its natural context and/or isrecombined with sequences that are not present in its natural context.

A “recombinant” polypeptide sequence is produced by translation from a“recombinant” polynucleotide sequence.

As used herein, the term “variant” refers to polynucleotide orpolypeptide sequences different from the specifically identifiedsequences, wherein one or more nucleotides or amino acid residues isdeleted, substituted, or added. Variants may be naturally occurringallelic variants, or non-naturally occurring variants. Variants may befrom the same or from other species and may encompass homologues,paralogues and orthologues. In certain embodiments, variants of thepolypeptides useful in the invention and biological activities that arethe same or similar to those of the parent polypeptides orpolynucleotides. The term “variant” with reference to polynucleotidesand polypeptides encompasses all forms of polynucleotides andpolypeptides as defined herein.

Variant polynucleotide sequences preferably exhibit at least 50%, atleast 60%, preferably at least 70%, preferably at least 71%, preferablyat least 72%, preferably at least 73%, preferably at least 74%,preferably at least 75%, preferably at least 76%, preferably at least77%, preferably at least 78%, preferably at least 79%, preferably atleast 80%, preferably at least 81%, preferably at least 82%, preferablyat least 83%, preferably at least 84%, preferably at least 85%,preferably at least 86%, preferably at least 87%, preferably at least88%, preferably at least 89%, preferably at least 90%, preferably atleast 91%, preferably at least 92%, preferably at least 93%, preferablyat least 94%, preferably at least 95%, preferably at least 96%,preferably at least 97%, preferably at least 98%, and preferably atleast 99% identity to a sequence of the present invention. Identity isfound over a comparison window of at least 10 nucleotide positions,preferably at least 15 nucleotide positions, preferably at least 20nucleotide positions, preferably at least 27 nucleotide positions,preferably at least 40 nucleotide positions, preferably at least 50nucleotide positions, and most preferably over the entire length of apolynucleotide of the invention.

Polynucleotide sequence identity may be calculated over the entirelength of the overlap between a candidate and subject polynucleotidesequences using global sequence alignment programs (e.g. Needleman, S.B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). A fullimplementation of the Needleman-Wunsch global alignment algorithm isfound in the needle program in the EMBOSS package (Rice, P. Longden, I.and Bleasby, A. EMBOSS: The European Molecular Biology Open SoftwareSuite, Trends in Genetics June 2000, vol 16, No 6. pp. 276-277) whichcan be obtained from http://www.hgmp.mre.ac.uk/Software/EMBOSS/. TheEuropean Bioinformatics Institute server also provides the facility toperform EMBOSS-needle global alignments between two sequences on line athttp:/www.ebi.ac.uk/emboss/align/.

Alternatively the GAP program may be used which computes an optimalglobal alignment of two sequences without penalizing terminal gaps. GAPis described in the following paper: Huang, X. (1994) On Global SequenceAlignment. Computer Applications in the Biosciences 10, 227-235.

Polynucleotide variants also encompass those which exhibit a similarityto one or more of the specifically identified sequences that is likelyto preserve the functional equivalence of those sequences and whichcould not reasonably be expected to have occurred by random chance. Thisprogram finds regions of similarity between the sequences and for eachsuch region reports an “E value” which is the expected number of timesone could expect to see such a match by chance in a database of a fixedreference size containing random sequences. The size of this database isset by default in the bl2seq program. For small E values, much less thanone, the E value is approximately the probability of such a randommatch.

Variant polynucleotide sequences preferably exhibit an E value of lessthan 1×10⁻⁵, more preferably less than 1×10⁻⁶, more preferably less than1×10⁻⁹, more preferably less than 1×10⁻¹², more preferably less than1×10⁻¹⁵, more preferably less than 1×10⁻¹⁸ and most preferably less than1×10⁻²¹ when compared with any one of the specifically identifiedsequences.

Use of BLASTN is preferred for use in the determination of sequenceidentity for polynucleotide variants according to the present invention.

The identity of polynucleotide sequences may be examined using thefollowing UNIX command line parameters:

bl2seq -i nucleotideseq1 -j nucleotideseq2 -F F -p blastn

The parameter -F F turns off filtering of low complexity sections. Theparameter -p selects the appropriate algorithm for the pair ofsequences. The bl2seq program reports sequence identity as both thenumber and percentage of identical nucleotides in a line “Identities=”.

Polynucleotide sequence identity and similarity can also be determinedin the following manner. The subject polynucleotide sequence is comparedto a candidate polynucleotide sequence using sequence alignmentalgorithms and sequence similarity search tools such as in Genbank,EMBL, Swiss-PROT and other databases. Nucleic Acids Res 29:1-10 and11-16, 2001 provides examples of online resources. BLASTN (from theBLAST suite of programs, version 2.2.13 Mar. 2007 in bl2seq (Tatiana A.et al, FEMS Microbiol Lett. 174:247-250 (1999), Altschul et al., Nuc.Acid Res 25:3389-3402, (1997)), which is publicly available from NCBI(ftp://ftp.ncbi.nih.gov/blast/) or from NCBI at Bethesda, Md., USA. Thedefault parameters of bl2seq are utilized except that filtering of lowcomplexity parts should be turned off.

Alternatively, variant polynucleotides hybridize to the specifiedpolynucleotide sequence, or a complement thereof under stringentconditions.

The term “hybridize under stringent conditions”, and grammaticalequivalents thereof, refers to the ability of a polynucleotide moleculeto hybridize to a target polynucleotide molecule (such as a targetpolynucleotide molecule immobilized on a DNA or RNA blot, such as aSouthern blot or Northern blot) under defined conditions of temperatureand salt concentration. The ability to hybridize under stringenthybridization conditions can be determined by initially hybridizingunder less stringent conditions then increasing the stringency to thedesired stringency.

With respect to polynucleotide molecules greater than about 100 bases inlength, typical stringent hybridization conditions are no more than 25to 30° C. (for example, 10° C.) below the melting temperature (Tm) ofthe native duplex (see generally, Sambrook et al., Eds, 1987, MolecularCloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubelet al., 1987, Current Protocols in Molecular Biology, Greene Publishing,incorporated herein by reference). Tm for polynucleotide moleculesgreater than about 100 bases can be calculated by the formulaTm=81.5+0.41% (G+C-log(Na+) (Sambrook et al., Eds, 1987, MolecularCloning, A Laboratory Manual, 2nd Ed. Gold Spring Harbor Press; Boltonand McCarthy, 1962, PNAS 84:1390). Typical stringent conditions for apolynucleotide of greater than 100 bases in length would behybridization conditions such as prewashing in a solution of 6×SSC, 0.2%SDS; hybridizing at 65° C., 6×SSC, 0.2% SDS overnight; followed by twowashes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of30 minutes each in 0.2×SSC, 0.1% SDS at 65° C.

In one embodiment stringent conditions use 50% formamide, 5×SSC, 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulphate at 42° C., with washes at 42° C. in 0.2×SSC and 50%formamide at 55° C., followed by a wash comprising of 0.1×SSC containingEDTA at 55° C.

With respect to polynucleotide molecules having a length less than 100bases, exemplary stringent hybridization conditions are 5 to 10° C.below Tm. On average, the Tm of a polynucleotide molecule of length lessthan 100 bp is reduced by approximately (500/oligonucleotide length)° C.

With respect to the DNA mimics known as peptide nucleic acids (PNAs)(Nielsen et al., Science. 1991 Dec. 6; 254(5037):1497-500) Tm values arehigher than those for DNA-DNA or DNA-RNA hybrids, and can be calculatedusing the formula described in Giesen et al., Nucleic Acids Res. 1998Nov. 1; 26(21):5004-6. Exemplary stringent hybridization conditions fora DNA-PNA hybrid having a length less than 100 bases are 5 to 10° C.below the Tm.

Variant polynucleotides also encompasses polynucleotides that differfrom the sequences of the invention but that, as a consequence of thedegeneracy of the genetic code, encode a polypeptide having similar,activity to a polypeptide encoded by a polynucleotide of the presentinvention. A sequence alteration that does not change the amino acidsequence of the polypeptide is a “silent variation”. Except for ATG(methionine) and TGG (tryptophan), other codons for the same amino acidmay be changed by art recognized techniques, e.g., to optimize codonexpression in a particular host organism.

Polynucleotide sequence alterations resulting in conservativesubstitutions of one or several amino acids in the encoded polypeptidesequence without significantly altering its biological activity are alsoincluded in the invention. A skilled artisan will be aware of methodsfor making phenotypically silent amino acid substitutions (see, e.g.,Bowie et al., 1990, Science 247, 1306).

Variant polynucleotides due to silent variations and conservativesubstitutions in the encoded polypeptide sequence may be determinedusing the bl2seq program via the tblastx algorithm as described above.

The term “variant” with reference to polypeptides also encompassesnaturally occurring, recombinantly and synthetically producedpolypeptides. Variant polypeptide sequences preferably exhibit at least50%, preferably at least 60%, preferably at least 70%, preferably atleast 71%, preferably at least 72%, preferably at least 73%, preferablyat least 74%, preferably at least 75%, preferably at least 76%,preferably at least 77%, preferably at least 78%, preferably at least79%, preferably at least 80%, preferably at least 81%, preferably atleast 82%, preferably at least 83%; preferably at least 84%, preferablyat least 85%, preferably at least 86%, preferably at least 87%,preferably at least 88%, preferably at least 89%, preferably at least90%, preferably at least 91%, preferably at least 92%, preferably atleast 93%, preferably at least 94%, preferably at least 95%, preferablyat least 96%, preferably at least 97%, preferably at least 98%, andpreferably at least 99% identity to a sequence of the present invention.Identity is found over a comparison window of at least 5 amino acidpositions, preferably at least 7 amino acid positions, preferably atleast 10 amino acid positions, preferably at least 15 amino acidpositions, preferably at least 20 amino acid positions and mostpreferably over the entire length of a polypeptide used in theinvention.

Polypeptide variants also encompass those which exhibit a similarity toone or more of the specifically identified sequences that is likely topreserve the functional equivalence of those sequences and which couldnot reasonably be expected to have occurred by random chance.

Polypeptide sequence identity and similarity can be determined in thefollowing manner. The subject polypeptide sequence is compared to acandidate polypeptide sequence using BLASTP (from the BLAST suite ofprograms, version 2.2.14 [May 2006]) in bl2seq, which is publiclyavailable from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The defaultparameters of bl2seq are utilized except that filtering of lowcomplexity regions should be turned off.

The similarity of polypeptide sequences may be examined using thefollowing UNIX command line parameters:

-   -   bl2seq peptideseq1 -j peptideseq2 -F F -p blastp

Variant polypeptide sequences preferably exhibit an E value of less than1×10⁻⁵, more preferably less than 1×10⁻⁶, more preferably less than1×10⁻⁹, more preferably less than 1×10⁻¹², more preferably less than1×10⁻¹⁵, more preferably less than 1×10⁻¹⁸ and most preferably less than1×10⁻²¹ when compared with any one of the specifically identifiedsequences.

The parameter -F F turns off filtering of low complexity sections. Theparameter -p selects the appropriate algorithm for the pair ofsequences. This program finds regions of similarity between thesequences and for each such region reports an “E value” which is theexpected number of times one could expect to see such a match by chancein a database of a fixed reference size containing random sequences. Forsmall &values, much less than one, this is approximately the probabilityof such a random match.

Polypeptide sequence identity may also be calculated over the entirelength of the overlap between a candidate and subject polypeptidesequences using global sequence alignment programs. EMBOSS-needle(available at http:/www.ebi.ac.uk/emboss/align/) and GAP (Huang, X.(1994) On Global Sequence Alignment. Computer Applications in theBiosciences 10, 227-235.) as discussed above are also suitable globalsequence alignment programs for calculating polypeptide sequenceidentity.

Use of BLASTP as described above is preferred for use in thedetermination of polypeptide variants according to the presentinvention.

Preferred variants include peptides who's sequence differs from thehuman BNP-SP (1-26) herein by one or more conservative amino acidsubstitutions, deletions, additions or insertions which do not affectthe biological activity of the peptide. Conservative substitutionstypically include the substitution of one amino acid for another withsimilar characteristics, e.g., substitutions within the followinggroups: valine, glycine; glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid; asparagines, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine. Example 8 of conservativesubstations can also be found in the sequences of BNP-SP in FIGS. 2A and2B whereby the substitutions in different mammalian species compared tothe human sequence are shown. Other conservative substitutions can betaken from Table 1 below.

TABLE 1 Exemplary Preferred Original Residue Substitutions SubstitutionAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; leu phe; norleucine Leu (L) norleucine; ile; ile val;met; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; leu tyr Pro (P) ala ala Ser (S) thr thr Thr (T)ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; leu ala; norleucine

Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg:

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for a member of another class. See for example R beingsubstituted with H at BNP-SP 25.

Other variants include peptides with modifications which influencepeptide stability. Such analogs may contain, for example, one or morenon-peptide bonds (which replace the peptide bonds) in the peptidesequence. Also included are analogs that include residues other thannaturally occurring L-amino acids, e.g. D-amino acids or non-naturallyoccurring synthetic amino acids, e.g. beta or gamma amino acids andcyclic analogs.

Substitutions, deletions, additions or insertions may be made bymutagenesis methods known in the art. A skilled worker will be aware ofmethods for making phenotypically silent amino acid substitutions. Seefor example Bowie et al., 1990, Science 247, 1306.¹⁰

Also included within the polypeptides of the invention are those whichhave been modified during or after synthesis for example bybiotinylation, benzylation, glycosylation, phosphorylation, amidation,by derivatization using blocking/protecting groups and the like. Suchmodifications may increase stability or activity of the polypeptide.

The term “genetic construct” refers to a polynucleotide molecule,usually double-stranded DNA, which may have inserted into it anotherpolynucleotide molecule (the insert polynucleotide molecule) such as,but not limited to, a cDNA molecule. A genetic construct may contain thenecessary elements that permit transcribing the insert polynucleotidemolecule, and, optionally, translating the transcript into apolypeptide. The insert polynucleotide molecule may be derived from thehost cell, or may be derived from a different cell or organism and/ormay be a recombinant polynucleotide. Once inside the host cell thegenetic construct may become integrated in the host chromosomal DNA. Thegenetic construct may be linked to a vector.

The term “vector” refers to a polynucleotide molecule, usually doublestranded DNA, which is used to transport the genetic construct into ahost cell. The vector may be capable of replication in at least oneadditional host system, such as E. coli.

The term “expression construct” refers to a genetic construct thatincludes the necessary elements that permit transcribing the insertpolynucleotide molecule, and, optionally, translating the transcriptinto a polypeptide. An expression construct typically comprises in a 5′to 3′ direction:

-   -   a) a promoter functional in the host cell into which the        construct will be transformed,    -   b) the polynucleotide to be expressed, and    -   c) a terminator functional in the host cell into which the        construct will be transformed.

The term “coding region” or “open reading frame” (ORF) refers to thesense strand of a genomic DNA sequence or a cDNA sequence that iscapable of producing a transcription product and/or a polypeptide underthe control of appropriate regulatory sequences. The coding sequence isidentified by the presence of a 5′ translation start codon and a 3′translation stop codon. When inserted into a genetic construct, a“coding sequence” is capable of being expressed when it is operablylinked to promoter and terminator sequences and/or other regulatoryelements.

“Operably-linked” means that the sequence to be expressed is placedunder the control of regulatory elements that include promoters,transcription control sequences, translation control sequences, originsof replication, tissue-specific regulatory elements, temporal regulatoryelements, enhancers, polyadenylation signals, repressors andterminators.

The term “noncoding region” refers to untranslated sequences that areupstream of the translational start site and downstream of thetranslational stop site. These sequences are also referred torespectively as the 5′ UTR and the 3′ UTR. These regions includeelements required for transcription initiation and termination and forregulation of translation efficiency.

Terminators are sequences, which terminate transcription, and are foundin the 3′ untranslated ends of genes downstream of the translatedsequence. Terminators are important determinants of mRNA stability andin some cases have been found to have spatial regulatory functions.

The term “promoter” refers to nontranscribed cis-regulatory elementsupstream of the coding region that regulate gene transcription.Promoters comprise cis-initiator elements which specify thetranscription initiation site and conserved boxes such as the TATA box,and motifs that are bound by transcription factors.

The terms “to alter expression of” and “altered expression” of apolynucleotide or polypeptide of the invention, are intended toencompass the situation where genomic DNA corresponding to apolynucleotide of the invention is modified thus leading to alteredexpression of a polynucleotide or polypeptide of the invention.Modification of the genomic DNA may be through genetic transformation orother methods known in the art for inducing mutations. The “alteredexpression” can be related to an increase or decrease in the amount ofmessenger RNA and/or polypeptide produced and may also result in alteredactivity of a polypeptide due to alterations in the sequence of apolynucleotide and polypeptide produced.

“Subject” as used herein is preferably a mammal and includes human, andnon-human mammals such as cats, dogs, horses, cows, sheep, deer, mice,rats, primates (including gorillas, rhesus monkeys and chimpanzees),possums and other domestic farm or zoo animals. Preferably, the mammalis human.

The term “presentation” as used herein refers to presentation of asubject at a medical facility such as a clinic or hospital.

The term “treat”, “treating” or “treatment” and “preventing” refer totherapeutic or prophylactic measures which alleviate, ameliorate,manage, prevent, restrain, stop or reverse progression of ACD, orcardiac transplant rejection or effects thereof, particularly of ACS.The subject may show observable or measurable (statisticallysignificant) reduction in one or more of TnI, BNP, N-BNP, and otherusual clinical markers known to those skilled in the art, indicatingimprovement.

It is intended that reference to a range of numbers disclosed herein(for example 1 to 10) also incorporates reference to all related numberswithin that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9and 10) and also any range of rational numbers within that range (forexample 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, allsub-ranges of all ranges expressly disclosed herein are expresslydisclosed. These are only examples of what is specifically intended andall possible combinations of numerical values between the lowest valueand the highest value enumerated are to be considered to be expresslystated in this application in a similar manner.

DETAILED DESCRIPTION OF THE INVENTION

Human B-type natriuretic peptide (BNP) is a member of the cardiacnatriuretic peptide family. As shown in FIG. 1, preproBNP is a 134 aminoacid molecule. The signal peptide BNP-SP (1-26) is cleaved to givepreproBNP (27-134). PreproBNP (27-134) is in turn further processed togive bioactive forms preproBNP (103-134) and prepro BNP (27-102). It islikely that BNP-SP is degraded into smaller fragments by signalpeptidase (SPP); usually near the hydrophobic central region of theBNP-SP (1-26) sequence.

It has long been thought that the functional role of the BNP-SP islimited to controlling the trafficking of BNP in the endoplasmicreticulum. Once this is achieved it has been assumed that the signalpeptide is then degraded without ever being secreted from the cell.

Very recently, it has been found that BNP-SP appears in the circulation(WO 2005/052593; US 2005/0244904). Based on this finding BNP-SP has beensuggested for use as a circulating biomarker for cardiac disease. Thepresent applicants have made a further and highly unexpected finding. Inpatients with acute myocardial infarction (AMI) the circulatingconcentration of BNP-SP is highest in the first few hours following theonset of the patient's symptoms—in fact, at the time of presentation tothe hospital or clinic. This is contrary to expectations that BNP-SPlevels would be correlated with N-BNP levels and could therefore beexpected to reach their peak 12 to 24 hours from onset of, or clinicalpresentation with ACD, cardiac transplant rejection, or with anundiagnosed or suspected ACD or pulmonary disorder. Levels observed inthe first few hours are surprisingly very high often reaching a peaksome four to ten, commonly five to eight fold higher than levels in anormal control population.

The level of BNP-SP remains up to three times higher than BNP-SP levelsin a control population for at least 6 weeks from first measurement onclinical presentation. These findings suggest BNP-SP is useful as a veryclear early stage marker of cardiac transplant rejection, ACD includingacute coronary syndromes (ACS) such as AMI, particularly non-ST elevatedMI, acute cardiac ischemia and may be used to distinguish ACD frompulmonary disorders.

Based on these surprising findings, the applicants have determined forthe first time, that it would be useful to screen for circulating BNP-SPor variants or fragments thereof, as well as nucleotide sequencesencoding BNP-SP or the variants and fragments thereof in a biologicalsample taken from a subject within two hours of onset of, or at clinicalpresentation with the disorder.

Useful in the invention are antigenic fragments or variants of BNP-SPwhich are least 5 amino acids in length. Particularly useful fragmentsare the N-terminus or C-terminus of BNP-SP. Examples of specificantigenic peptides are BNP-SP (1-10) (SEQ ID NO:13), BNP-SP (1-17) (SEQID NO:15), BNP-SP (12-23) (SEQ ID NO:17), and BNP-SP (17-26) (SEQ IDNO:19). Corresponding nucleotide sequences are given in SEQ ID NOs: 14,16, 18 and 20 respectively. These sequences are provided by theapplicants for the first time. Both the nucleic acid molecules andpeptides form aspects of the invention.

Accordingly, in a first aspect, the invention provides a nucleic acidmolecule encoding a BNP-SP of the invention selected from

-   -   (a) SEQ ID NO:14;    -   (b) SEQ ID NO:16;    -   (c) SEQ ID NO:18;    -   (d) SEQ ID NO:20;    -   (e) a complement of any one of (a) to (d);    -   (f) a sequence of at least 15 nucleotides in length, capable of        hybridising to the sequence of any one of (a) to (e) under        stringent conditions with the proviso that the sequence is not        ccagtgcacaagctgatggggaggcgaga or SEQ ID NO: 22.

The invention also provides isolated BNP-SP polypeptides encoded by anucleic acid molecule of the invention.

Specific polypeptides of the invention include polypeptides having theamino acid sequences of SEQ ID NOs: 13, 15, 17 and 19 all as set forthin the accompanying sequence listing. Also contemplated are functionallyequivalent variants and fragments of these polypeptides as definedherein.

The nucleic acid molecules of the invention or otherwise describedherein are preferably isolated. They can be isolated from a biologicalsample using a variety of techniques known to those of ordinary skill inthe art. By way of example, such polynucleotides can be isolated throughuse of the polymerase chain reaction (PCR) described in Mullis et al.,Eds. 1994 The Polymerase Chain Reaction, Birkhauser. The nucleic acidmolecules of the invention can be amplified using primers, as definedherein, derived from the polynucleotide sequences of the invention.

Further methods for isolating polynucleotides include use of all, orportions of, the polynucleotide of the invention, particularly apolynucleotide having the sequence set forth in SEQ ID NO:19 ashybridization probes. The technique of hybridizing labeledpolynucleotide probes to polynucleotides immobilized on solid supportssuch as nitrocellulose filters or nylon membranes, can be used to screengenomic or cDNA libraries. Similarly, probes may be coupled to beads andhybridized to the target sequence. Isolation can be effected using knownart protocols such as magnetic separation. Exemplary stringenthybridization and wash conditions are as given above.

Polynucleotide fragments may be produced by techniques well-known in theart such as restriction endonuclease digestion and oligonucleotidesynthesis.

A partial polynucleotide sequence may be used as a probe, in methodswell-known in the art to identify the corresponding full lengthpolynucleotide sequence in a sample. Such methods include PCR-basedmethods, 5′RACE (Methods Enzymol. 218: 340-56 (1993); Sambrook et al.,Supra) and hybridization-based method, computer/database-based methods.Detectable labels such as radioisotopes, fluorescent, chemiluminescentand bioluminescent labels may be used to facilitate detection. InversePCR also permits acquisition of unknown sequences, flanking thepolynucleotide sequences disclosed herein, starting with primers basedon a known region (Triglia et al., Nucleic Acids Res 16, 8186, (1998))The method uses several restriction enzymes to generate a suitablefragment in the known region of a gene. The fragment is thencircularized by intramolecular ligation and used as a PCR template.Divergent primers are designed from the known region. In order tophysically assemble full-length clones, standard molecular biologyapproaches can be utilized (Sambrook et al., Supra). Primers and primerpairs which allow amplification of polynucleotides of the invention,also form a further aspect of this invention.

Variants (including orthologues) may be identified by the methodsdescribed. Variant polynucleotides may be identified using PCR-basedmethods (Mullis et al., Eds. 1994 The Polymerase Chain Reaction,Birkhauser). Typically, the polynucleotide sequence of a primer, usefulto amplify variants of polynucleotide molecules by PCR, may be based ona sequence encoding a conserved region of the corresponding amino acidsequence.

Further methods for identifying variant polynucleotides include use ofall, or portions of, the specified polynucleotides as hybridizationprobes to screen genomic or cDNA libraries as described above. Typicallyprobes based on a sequence encoding a conserved region of thecorresponding amino acid sequence may be used. Hybridisation conditionsmay also be less stringent than those used when screening for sequencesidentical to the probe.

The variant sequences, including both polynucleotide and polypeptidevariants, may also be identified by the computer-based methods discussedabove.

Multiple sequence alignments of a group of related sequences can becarried out with CLUSTALW (Thompson, et al., Nucleic Acids Research,22:4673-4680 (1994),http://www-igbmc.u-strasbg.fr/BioInfo/ClustalW/Top.html) or T-COFFEE(Cedric Notredame et al., J. Mol. Biol. 302: 205-217 (2000))) or PILEUP,which uses progressive, pairwise alignments. (Feng et al., J. Mol. Evol.25, 351 (1987)).

Pattern recognition software applications are available for findingmotifs or signature sequences. For example, MEME (Multiple Em for MotifElicitation) finds motifs and signature sequences in a set of sequences,and MAST (Motif Alignment and Search Tool) uses these motifs to identifysimilar or the same motifs in query sequences. The MAST results areprovided as a series of alignments with appropriate statistical data anda visual overview of the motifs found. MEME and MAST were developed atthe University of California, San Diego.

PROSITE (Bairoch et al., Nucleic Acids Res. 22, 3583 (1994); Hofmann etal., Nucleic Acids Res. 27, 215 (1999)) is a method of identifying thefunctions of uncharacterized proteins translated from genomic or cDNAsequences. The PROSITE database (www.expasy.org/prosite) containsbiologically significant patterns and profiles and is designed so thatit can be used with appropriate computational tools to assign a newsequence to a known family of proteins or to determine which knowndomain(s) are present in the sequence (Falquet et al., Nucleic AcidsRes. 30, 235 (2002)). Prosearch is a tool that can search SWISS-PROT andEMBL databases with a given sequence pattern or signature.

Proteins can be classified according to their sequence relatedness toother proteins in the same genome (paralogues) or a different genome(orthologues). Orthologous genes are genes that evolved by speciationfrom a common ancestral gene and normally retain the same function asthey evolve. Paralogous genes are genes that are duplicated within agenome and genes may acquire new specificities or modified functionswhich may be related to the original one. Phylogenetic analysis methodsare reviewed in Tatusov et al., Science 278, 631-637, 1997).

In addition to the computer/database methods described above,polypeptide variants may be identified by physical methods, for exampleby screening expression libraries using antibodies raised againstpolypeptides of the invention (Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) byrecombinant DNA techniques also described by Sambrook et al. or byidentifying polypeptides from natural sources with the aid of suchantibodies.

Polypeptides, including variant polypeptides, may be prepared usingpeptide synthesis methods well known in the art such as direct peptidesynthesis using solid phase techniques (e.g. Merrifield, 1963, in J. Am.Chem. Soc. 85, 2149; Stewart et al., 1969, in Solid-Phase PeptideSynthesis, WH Freeman Co, San Francisco Calif.; Matteucci et al. J. Am.Chem. Soc. 103:3185-3191, 1981) or automated synthesis, for exampleusing a Synthesiser from Applied Biosystems (California, USA). Mutatedforms of the polypeptides may also be produced using synthetic methodssuch as site-specific mutagensis of the DNA encoding the amino acidsequence as described by Adelmen et al; DNA 2, 183 (1983).

The polypeptides and variant polypeptides herein are preferablyisolated. They may be isolated or purified from natural sources using avariety of techniques that are well known in the art (e.g. Deutscher,1990, Ed, Methods in Enzymology, Vol. 182, Guide to ProteinPurification). Technologies include HPLC, ion-exchange chromatography,and immunochromatography but are not limited thereto.

Alternatively the polypeptides and variant polypeptides may be expressedrecombinantly in suitable host cells and separated from the cells asdiscussed below. The polypeptides and variants have utility ingenerating antibodies, and generating ligands amongst other uses.

The genetic constructs described herein may comprise one or more of thedisclosed polynucleotide sequences and/or polynucleotides encoding thedisclosed polypeptides, of the invention and may be useful fortransforming, for example, bacterial, fungal, insect, mammalian or plantorganisms. The genetic constructs of the invention are intended toinclude expression constructs as herein defined. Included are vectors(such as pBR322, pUC18, pU19, Mp18, Mp19, ColE1, PCR1 and pKRC), phages(such as lambda gt10), and M13 plasmids (such as pBR322, pACYC184,pT127, RP4, p13101, SV40 and BPV), cosmids, YACS, BACs shuttle vectorssuch as pSA3, PAT28 transposons (such as described in U.S. Pat. No.5,792,294) and the like.

The constructs may conveniently include a selection gene or selectablemarker. Typically an antibiotic resistance marker such as ampicillin,methotrexate, or tetracycline is used.

Promoters useful in the constructs include β-lactamase, alkalinephosphatase, tryptophan, and tac promoter systems which are all wellknown in the art. Yeast promoters include 3-phosphoglycerate kinase,enolase, hexokinase, pyruvate decarboxylase, glucokinase, andglyceraldehydrate-3-phosphanate dehydrogenase but are not limitedthereto.

Enhancers may also be employed to act on the promoters to enhancetranscription. Suitable enhancers for use herein include SV40 enhancer,cytomeglovirus early promoter enhancer, globin, albumin, insulin and thelike.

Methods for producing and using genetic constructs and vectors are wellknown in the art and are described generally in Sambrook et al.,(supra), and Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing, 0.1987. Methods for transforming selected host cellswith the vectors are also known, for example, the calcium chloridetreatment described by Cohen, S N; PNAS 69, 2110, 1972.

Host cells comprising the genetic constructs and vectors described maybe derived from prokaryotic or eukaryotic sources, for example yeast,bacteria, fungi, insect (eg baculovirus), animal, mammalian or plantorganisms. In one embodiment the host cells are isolated host cells.Prokaryotes most commonly employed as host cells are strains of E. coli.Other prokaryotic hosts include Pseudomonas, Bacillus, Serratia,Klebsiella, Streptomyces, Listeria, Saccharomyces, Salmonella andMycobacteria but are not limited thereto.

Eukaryotic cells for expression of recombinant protein include butarenot limited to Vero cells, HeLa, CHO (Chinese Hamster ovary cells),293, BHK cells, MDCK cells, and COS cells as well as prostate cancercell lines such as PrEC, LNCaP, Du 145 and RWPE-2. The cells areavailable from ATCC, Virginia, USA.

Prokaryotic promoters compatible with expression of nucleic acidmolecules of the invention include known art constitutive promoters(such as the int promoter of bacteriophage lamda and the bla promoter ofthe beta-lactamase gene sequence of pBR322) and regulatable promoters(such as lacZ, recA and gal). A ribosome binding site upstream of thecoding sequence may also be required for expression.

Host cells comprising genetic constructs, such as expression constructs,are useful in methods for recombinant production of polypeptides. Suchmethods are well known in the art (see for example Sambrook et al.supra). The methods commonly involve the culture of host cells in anappropriate medium in conditions suitable for or conducive to,expression and selection of a polypeptide of the invention. Cells with aselectable marker may additionally be grown on medium appropriate forselection of host cells expressing a polypeptide of the invention.Transformed host cells expressing a polypeptide of the invention areselected and cultured under conditions suitable for expression of thepolypeptide. The expressed recombinant polypeptide, may be separated andpurified from the culture medium using methods well known in the artincluding ammonium sulfate precipitation, ion exchange chromatography,gel filtration, affinity chromatography, electrophoresis and the like(e.g. Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide toProtein Purification). Host cells may also be useful in methods forproduction of a product generated by an expressed polypeptide of theinvention.

In another aspect, the present invention provides a method forpredicting, diagnosing or monitoring an acute cardiac disorder (ACD) ina subject, the method comprising: measuring the level of BNP-SP in abiological sample obtained from the subject within two hours of onset ofthe ACD, or within two hours of presentation with ACD; and comparing thelevel of said BNP-SP with the BNP-SP level from a control wherein ameasured level of BNP-SP higher than the control level is indicative ofACD.

In another aspect the invention provides a method for monitoring aresponse to treatment of a an acute cardiac disorder (ACD) in a subject,the method comprising measuring the level of BNP-SP in a biologicalsample obtained from the subject within two hours of onset of the ACD,or within two hours of presentation with the ACD; and comparing thelevel of said BNP-SP with the BNP-SP level from a control, wherein achange in the measured level of BNP-SP from the control level isindicative of a response to the treatment.

It is known in the art that BNP precursors such as proBNP27-102proBNP27-47, can be used in predicting or diagnosing a cardiactransplant rejection episode and to distinguish between pulmonary andcardiovascular causes of dyspnea (shortness of breath). See US2005/0244902. Accordingly, it is similarly predictable that BNP-SP canbe used as an early marker of cardiac transplant rejection based oncardiac tissue analysis, and to distinguish pulmonary from acute cardiacdisorders.

Accordingly, the invention also provides a method for predicting,diagnosing or monitoring a cardiac transplant rejection episode in asubject, the method comprising measuring the level of BNP-SP in abiological sample obtained from a subject within two hours of hearttransplant and comparing the level of said BNP-SP with the BNP-SP levelfrom a control, wherein a measured level of BNP-SP higher than thecontrol level is indicative of transplant rejection.

The invention also provides a method of distinguishing between apulmonary disorder and an acute cardiac disorder (ACD) in a subject, themethod comprising measuring the level of BNP-SP in a biological sampleobtained from a subject within two hours of presentation with thedisorder; and comparing the level of said BNP-SP with the BNP-SP levelfrom a control wherein a measured BNP-SP level higher than the controllevel is indicative of ACD.

In one embodiment, the invention provides a method for predicting,diagnosing or monitoring an acute cardiac disorder (ACD), cardiactransplant rejection, or ACD/pulmonary disorder in a subject, the methodcomprising measuring the level of BNP-SP in a biological sample obtainedfrom the subject within the first two hours of onset of, or clinicalpresentation with ACD, transplant rejection or ACD/pulmonary disorder.

Preferably, the measured level of BNP-SP is compared with the BNP-SPlevel from a control wherein a measured level of BNP-SP higher than thecontrol level is indicative of ACD or transplant rejection.

The skilled reader will appreciate that for evaluation purposes, markerrequires correlation with a reference valve or control value.

As used herein a control can be an individual or group from which BNP-SPsamples are taken and a mean BNP-SP level determined. Usually, theindividual or group will comprise normal healthy individuals or a groupof individuals not known to be suffering from ACD, cardiac transplantrejection or ACD/pulmonary disorder. BNP-SP levels in most individualsare between 0-15 pmol/L, and the mean control level is about 10 pmol/L.Alternatively, the control level may be assessed based on a plurality ofreadings from previously tested individuals or groups. Another exampleof a control level is a ratiometric measure between BNP-SP and BNPlevels in cardiac tissue. The subject's BNP-SP level can be compared tothe mean BNP-SP level for that control population. The BNP-SP level inthe cardiac control population may be in the order of 1.5 to 3, commonly2 to 3 or 2.5 to 3 times higher than BNP-SP levels in the normal controlpopulation. Alternatively, the control may be one or more readings orthe mean of such readings taken from the same subject at an earliertime. Ascertaining appropriate controls and control levels forparticular methods is well known in the art.

The term within two hours of onset or clinical presentation includesfrom 1 minute up to and including 120 minutes from onset of, orpresentation at a medical facility with ACD, cardiac transplantrejection or an undiagnosed or suspected ACD/pulmonary disorder.Preferably measurements are made within 1 hour (from 1 minute up to andincluding 60 minutes) from onset or presentation, preferably within 5 to45 minutes, preferably 15 to 40 minutes, preferably 20 to 35 minutes,and optimally within 25 to 30 minutes of onset or presentation.

A level “higher” than a control, or a change or deviation from a controlis preferably statistically significant. A higher level, deviation from,or change from a control level or mean control level can be consideredto exist if the level differs from the control level by 5% or more, by10% or more, preferably by 20% ore more, more preferably by 50% or morecompared to the control level. Statistically significant mayalternatively be calculated as P≦0.05. In a further alternative, higherlevels, deviation and changes can be determined by recourse to assayreference limits or reference intervals. These can be calculated fromintuitive assessment or non-parametric methods. Overall, these methodscalculate the 0.025, and 0.975 fractiles as 0.025*(n+1) and 0.975 (n+1).Such methods are well known in the art.^(23,24) Presence of a markerabsent in a control, is also contemplated as a higher level, deviationor change.

It will be appreciated that the step of measuring BNP-SP levels in asample may be a single measurement on a single sample, or repeatedmeasurements on a number of samples. Accordingly, measurement maycomprise 1 to 20 measurements of BNP-SP, preferably 1 to 10, preferably1 to 5, preferably 1 to 3, preferably 1 or 2, preferably 2 or 3measurements, in samples taken at different times within the first twohours, preferably within one hour of, onset of or clinical presentation.Single, or repeated measurements outside the two hour period may also betaken to establish whether the BNP-SP level has fallen to the normalcontrol level, or cardiac control level.

In one preferred embodiment, the method comprises measuring BNP-SPlevels in 1 or 2 samples taken within the first hour of onset orpresentation, followed by measuring BNP-SP levels in 1 or 2 samplestaken within two to four hours of onset or presentation, or initialmeasurement of the BNP-SP level, preferably within two to three hours.

As noted above, BNP-SP levels measured within the first two hours ofonset or presentation are usually four to ten times higher, commonlyfive to eight times higher than BNP-SP levels measured in a normalcontrol. As stated above, also included within the ranges are thespecific ranges 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9,5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to9, 7 to 8, 8 to 10, 8 to 9, and 9 to 10 times.

In another embodiment, a level of BNP-SP in the sample in the range 20to 300 pmol/L, preferably 25 to 250 pmol/L, preferably 30 to 180 pmol/L,preferably 35 to 150 pmol/L, preferably 40 to 120 pmol/L, preferably 40to 90 pmol/L, and preferably 45 to 80 pmol/L is indicative of ACD,cardiac transplant rejection, or distinguishes ACD from a pulmonarydisorder.

As stated above, the ranges also include any values within the rangesuch as 20 to 180 pmol/L, 50 to 200 pmol/L, 40 to 130 pmol/L, 50 to 100pmol/L, 45 to 160 pmol/L, and the like.

The biological sample can be any biological material in which BNP-SP canbe located or secreted. This includes any tissue or bodily fluid such asblood, saliva, interstitial fluid, serum, plasma, urine, pericardialfluid and cerebrospinal fluid but is not limited thereto. Preferably thebiological sample is a circulatory biological sample, for example blood,serum or plasma. In one embodiment, the biological sample is cardiactissue.

The presence of the markers and their level of expression in the samplemay be determined according to methods known in the art such as SouthernBlotting, Northern Blotting, FISH or quantative PCR to quantitate thetranscription of mRNA [(Thomas, Pro. NAH, Acad. Sci. USA 77: 5201-52051980), (Jain K K., Med Device Technol. 2004 May; 15(4):14-7)], dotblotting, (DNA analysis) or in situ hybridization using an appropriatelylabelled probe, based on the marker sequences provided herein.

Accordingly, the invention also provides an assay for detecting thepresence of a nucleic acid molecule of the invention, in a sample, themethod comprising:

-   (a) contacting the sample with a polynucleotide probe which    hybridises to the nucleic acid sequence under stringent    hybridisation conditions; and-   (b) detecting the presence of a hybridisation complex in the sample.

Preferably the hybridisation probe is a labelled probe. Examples oflabels include fluorescent, chemiluminescent, radioenzyme andbiotin-avidin labels. Labelling and visualisation of labelled probes iscarried out according to known art methods such as those above.

For convenience the nucleic acid probe may be immobilized on a solidsupport including resins (such as polyacrylamides), carbohydrates (suchas sepharose), plastics (such as polycarbonate), and latex beads.

As discussed above the nucleic acid molecule probe may preferably be anRNA, cDNA or DNA molecule. Preferred probes include SEQ ID NOs: 14, 16,18, 20 and 22.

Stringent hybridisation conditions are as discussed above.

The expression level of the nucleic acid marker may be determined usingknown art techniques such as RT-PCR and electrophoresis techniquesincluding SDS-PAGE. Using these techniques the DNA or cDNA sequence of anucleic acid molecule of the invention, in a subject sample isamplified, and the level of DNA or cDNA or RNA measured.

In an alternate method the DNA, cDNA or RNA level may be measureddirectly in the sample without amplification.

A currently preferred method is Northern blot hybridization analysis.Probes for use in Northern blot hybridization analysis may be preparedbased on the marker sequences identified herein. A probe preferablyincludes at least 12, at least 15, at least 18, at least 24, at least30, at least 36, preferably at least 42, preferably at least 51,preferably at least 60, preferably at least 70 or more contiguousnucleotides of a reference sequence.

Alternatively, the expression level may be measured using reversetranscription based PCR(RT-PCR) assays using primers specific for thenucleic acid sequences. If desired, comparison of the level of themarker in the sample can be made with reference to a control nucleicacid molecule the expression of which is independent of the parameter orcondition being measured. A control nucleic acid molecule refers to amolecule in which the level does not differ between the disorder ortransplant rejection state and the healthy state. Levels of the controlmolecule can be used to normalise levels in the compared populations. Anexample of such a control molecule is GAP-DH. The markers of theinvention will change levels with the disorder.

In one embodiment the measuring step comprises detecting binding betweenBNP-SP and a binding agent that selectively binds BNP-SP or a fragmentor variant thereof. Preferably, the binding agent has lowcross-reactivity with other markers of biological events, and moreparticularly BNP or NT-BNP. The binding agent is preferably an antibodyor fragment thereof.

The present invention also relates to such antibodies, or fragments ofthe antibodies. An antibody that binds to BNP-SP or a fragment orvariant thereof may be in any form, including all classes of polyclonal,monoclonal, single chain, human, humanized antibodies and chimericantibodies produced by genetic recombination. Also included is antiserumobtained by immunizing an animal such as a mouse, rat or rabbit withBNP-SP or a fragment or variant thereof.

A fragment of an antibody or a modified antibody may also be used hereinso long as it binds BNP-SP or a fragment or variant thereof. Theantibody fragment may be Fab, F(ab′), F(ab′), and Fc or Fv fragment orsingle chain Fv (scFv), in which Fv fragments from H and L chains areligated by an appropriate linker (Huston et al. Proc. Natl. Acad. Sci.USA 85:5879-83 (1988)). The “Fc” portion of an antibody refers to thatportion of an immunoglobulin heavy chain that comprises one or moreheavy chain constant region domains; CH1, CH2 and CH3, but does notinclude the heavy chain variable region.

Methods for preparing antibodies are well known in the art (see forexample Harlow and Lane (1998).¹¹ Most commonly used antibodies areproduced by immunizing a suitable host mammal. Fusion proteinscomprising BNP-SP may also be used as immunogens.

An antibody may be modified by conjugation with a variety of molecules,such as polyethylene glycol (PEG). The modified antibody can be obtainedby chemically modifying an antibody. These modification methods areconventional in the field.

Alternatively, an antibody may be obtained as a chimeric antibody,between a variable region derived from nonhuman antibody and theconstant region derived from human antibody, or as a humanized antibody,comprising the complementarity determining region (CDR) derived fromnonhuman antibody, the frame work region (FR) derived from humanantibody, and the constant region. Such antibodies can be prepared usingknown art methods.

In brief, methods of preparing polyclonal antibodies are known to theskilled artisan. Polyclonal antibodies can be raised in a mammal, forexample, by one or more injections of an immunizing agent and, ifdesired, an adjuvant. Typically, the immunizing agent and/or adjuvantwill be injected in the mammal by multiple subcutaneous orintraperitoneal injections. The immunizing agent may include BNP-SP or afragment or variant thereof or a fusion protein thereof. It may beuseful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized. Examples of such immunogenicproteins include but are not limited to keyhole limpet hemocyanin,bovine serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

Monoclonal antibodies may be prepared using hybridoma methods well knownin the art. See for example Kohler and Milstein, 1975¹² and U.S. Pat.No. 4,196,265. The hybridoma cells may be cultured in a suitable culturemedium, alternatively, the hybridoma cells may be grown in vivo asascites in a mammal. Preferred immortalized cell lines are murinemyeloma lines, which can be obtained, for example, from the AmericanType Culture Collection, Virginia, USA. Immunoassays may be used toscreen for immortalized cell lines which secrete the antibody ofinterest. Sequences of BNP-SP or fragments or variants thereof may beused in screening.

Accordingly, also contemplated herein ate hybridomas which areimmortalized cell lines capable of secreting a BNP-SP specificmonoclonal antibody.

Well known means for establishing binding specificity of monoclonalantibodies produced by the hybridoma cells include immunoprecipitation,radiolinked immunoassay (MA), enzyme-linked immunoabsorbent assay(ELISA) and Western blot. (Lutz et al., Exp. Cell. Res. 175:109-124(1988)). Samples from immunised animals may similarly be screened forthe presence of polyclonal antibodies.

To facilitate detection, antibodies and fragments herein may be labelledwith detectable markers such as fluorescent, bioluminescent, andchemiluminescent compounds, as well as radioisotopes, magnetic beads andaffinity labels (e.g biotin and avidin). Examples of labels which permitindirect measurement of binding include enzymes where the substrate mayprovide for a coloured fluorescent product, suitable enzymes includehorseradish peroxidase, alkaline phosphatase, malate dehydrogenase andthe like. Fluorochromes (e.g Texas Red, fluorescein, phycobiliproteins,and phycoerythrin) can be used with a fluorescence activated cellsorter. Labelling techniques are well known in the art.

The monoclonal antibodies secreted by the cells may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxyapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies or fragments may also be produced byrecombinant DNA means (see for example U.S. Pat. No. 4,816,567). DNAmodifications such as substituting the coding sequence for human heavyand light chain constant domains in place of the homologous murinesequences (U.S. Pat. No. 4,816,567 above) are also possible. Theantibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. Production of chimericbivalent antibodies are also contemplated herein.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized antibodies include humanimmunoglobulins in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species. The production of humanized antibodies from non-humansources such as rabbit, rat and mouse are well known.^(13,14,15)

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries¹⁶; and transgenic methods,see, for example Neuberger 1996¹⁷; and Vaughan et al, 1998¹⁸.

Bispecific antibodies may also be useful. These antibodies aremonoclonal, preferably human or humanized, antibodies that have bindingspecificities for at least two different antigens. For example BNP-SP ora variant or fragment thereof, and an antigen selected from the groupincluding preproBNP, BNP, CK-MB, TnT, TnI, and myoglobin. Antibodieswith greater than two specificities for example trispecific antibodiesare also contemplated herein.

Methods for making bispecific antibodies are known in the art. See forexample Milstein and Cuello 1983¹⁹, Suresh et al., 1986²⁰ and Brennan etal., 1985²¹.

The BNP-SP which is selectively bound by the antibody is BNP-SP or anantigenic variant or fragment thereof as discussed above.

Desirably, the antibody binds the N-terminus or C-terminus of BNP-SP.Examples of specific antigenic peptides which the binding agentselectively binds include BNP-SP(1-10) SEQ ID NO:13, BNP-SP (1-17) SEQID NO:15, BNP-SP (12-23) (SEQ ID NO:17), BNP-SP(17-26) SEQ ID NO:19, andBNP-SP(1-26) SEQ ID NO:21.

Binding of BNP-SP can be detected by any means known in the artincluding specific (antibody based) and non specific (such as HPLC solidphase). Most commonly, antibodies herein are detected using an assaysuch as ELISA or RIA as noted above. Competitive binding assays,sandwich assays, non-competitive assays, fluoroimmunoassay,immunofluorometric assay, or immunoradiometric assays, luminescenceassays, chemiluniescence assays and mass spectrometry analysis such asurface-enhanced laser desorption and ionization (SELDI) electrosprayionization (ESI), matrix assisted laser-desorption ionization (MALDI),fourier transform Ion cyclotron resonance mass spectroscopy (FTICR)alone or in combination with non-specific binding agents such aschromatography formats are also feasible.

Conveniently, an antibody can be fixed to a solid substrate tofacilitate washing and isolation of the BNP-SP/antibody complex. Bindingof antibodies to a solid support can be achieved using known arttechniques. See for example Handbook of Experimental Immunology, 4thedition, Blackwell Scientific Publications, Oxford (1986). Useful solidsubstrates for antibodies include glass, nylon, paper and plastics.Similarly, BNP-SP can be adsorbed onto a solid substrate such asadsorbent silica, or resin particles, or silicon chips optionally coatedor derivatised with ion exchange, reverse phase (eg C18 coating) orother materials. The substrate may be in the form of beads, plates,tubes, sticks or biochips. Biochips or plates with addressable locationsand discreet microtitre plates are particularly useful. Also preferredfor use are multiple systems where beads containing antibodies directedto multiple analytes are used to measure levels of the analytes in asingle sample. Analytes to be measured may include other cardiac markersas well as BNP-SP or variants or fragments thereof. One example of asuitable multiplex bead system for use herein is the Luminex FluorokineMultianalyte Profiling system.

Antibody assay methods are well known in the art see for example U.S.Pat. No. 5,221,685, U.S. Pat. No. 5,310,687, U.S. Pat. No. 5,480,792,U.S. Pat. No. 5,525,524, U.S. Pat. No. 5,679,526, U.S. Pat. No.5,824,799, U.S. Pat. No. 5,851,776, U.S. Pat. No. 5,885,527, U.S. Pat.No. 5,922,615, U.S. Pat. No. 5,939,272, U.S. Pat. No. 5,647,124, U.S.Pat. No. 5,985,579, U.S. Pat. No. 6,019,944, U.S. Pat. No. 6,113,855,U.S. Pat. No. 6,143,576 and for unlabelled assays U.S. Pat. No.5,955,377, and U.S. Pat. No. 5,631,171 see also Zola, MonoclonalAntibodies: A Manual of Techniques pp 147-158 (CRC Press, Inc 1987),Harlow and Lane (1998) Antibodies, A Laboratory Manual, Cold SpringHarbour Publications, New York, and US 2005/0064511 for a description ofassay formats and conditions all of the above references areincorporated herein by reference in their entirety.

Immunoassay analysers are also well known and include Beckman Acess,Abbott AxSym, Roche ElecSys and Dade Behring Status systems amongstothers which are well described²².

Binding of BNP-SP and an antibody to form a complex can be detecteddirectly or indirectly. Direct detection is carried out using labelssuch as fluorescence, luminescence, radionuclides, metals, dyes and thelike. Indirect detection includes binding detectable labels such asdigoxin or enzymes such as horseradish peroxidase and alkalinephosphatase to form a labelled BNP-SP antibody followed by a step ofdetecting the label by addition of detection reagents.

Horseradish peroxidase for example can be incubated with substrates suchas o-Phenylenediamine Dihyhydrochloride (OPD) and peroxide to generate acoloured product whose absorbance can be measured, or with luminol andperoxide to give chemiluminescent light which can be measured in aluminometer as is known in the art. Biotin or digoxin can be reactedwith binding agents that bind strongly to them. For example, theproteins avidin and streptavidin will bind strongly to biotin. A furthermeasurable label is then covalently bound or linked thereto either bydirect reaction with the protein, or through the use of commonlyavailable crosslinking agents such as MCS and carbodiimide, or byaddition of chelating agents.

Generally, the complex is separated from the uncomplexed reagents forexample by centrifugation. If the antibody is labelled, the amount ofcomplex will be reflected by the amount of label detected.Alternatively, a BNP-SP may be labelled by binding to an, antibody anddetected in a competitive assay by measuring a reduction in boundlabelled BNP-SP when the antibody-labelled-BNP-SP is incubated with abiological sample containing unlabelled BNP-SP. Other immunoassays maybe used for example a sandwich assay.

In one example, following contact with the antibody, usually overnightfor 18 to 25 hours at 4° C., or for 1 to 2 to 4 hours at 25° C. to 40°C., the labelled BNP-SP bound to the binding agent (antibody) isseparated from the unbound labelled BNP-SP. In solution phase assays,the separation may be accomplished by addition of an anti gamma globulinantibody (second-antibody) coupled to solid phase particles such ascellulose, or magnetic material. The second-antibody is raised in adifferent species to that used for the primary antibody and binds theprimary antibody. All primary antibodies are therefore bound to thesolid phase via the second antibody. This complex is removed fromsolution by centrifugation or magnetic attraction and the bound labelledpeptide measured using the label bound to it. Other options forseparating bound from free label include formation of immune complexes,which precipitate from solution, precipitation of the antibodies bypolyethyleneglycol or binding free labelled peptide to charcoal andremoval from solution by centrifugation of filtration. The label in theseparated bound or free phase is measured by an appropriate method suchas those presented above.

Competitive binding assays can also be configured as solid phase assaysthat are easier to perform and are therefore preferable to those above.This type of assay uses plates with wells (commonly known as ELISA orimmunoassay plates), solid beads or the surfaces of tubes. The primaryantibody is either adsorbed or covalently bound to the surface of theplate, bead or tube, or is bound indirectly through a second anti gammaglobulin or anti Fc region antibody adsorbed or covalently bound to theplate. Sample and labelled peptide (as above) are added to the plateeither together or sequentially and incubated under conditions allowingcompetition for antibody binding between BNP-SP in the sample and thelabelled peptide. Unbound labelled peptide can subsequently be aspiratedoff and the plate rinsed leaving the antibody bound labelled peptideattached to the plate. The labelled peptide can then be measured usingtechniques described above.

Sandwich type assays are more preferred for reasons of specificity,speed and greater measuring range. In this type of assay an excess ofthe primary antibody to BNP-SP is attached to the well of an ELISAplate, bead or tube via adsorption, covalent coupling, or an anti Fc orgamma globulin antibody, as described above for solid phase competitionbinding assays. Sample fluid or extract is contacted with the antibodyattached to the solid phase. Because the antibody is in excess thisbinding reaction is usually rapid. A second antibody to BNP-SP is alsoincubated with the sample either simultaneously or sequentially with theprimary antibody. This second antibody is chosen to bind to a site onBNP-SP that is different from the binding site of the primary antibody.These two antibody reactions result in a sandwich with the BNP-SP fromthe sample sandwiched between the two antibodies. The second antibody isusually labelled with a readily measurable compound as detailed abovefor competitive binding assays. Alternatively a labelled third antibodywhich binds specifically to the second antibody may be contacted withthe sample. After washing away the unbound material the bound labelledantibody can be measured and quantified by methods outlined forcompetitive binding assays.

A dipstick type assay may also be used. These assays are well known inthe art. They may for example, employ small particles such as gold orcoloured latex particles with specific antibodies attached. The liquidsample to be measured may be added to one end of a membrane or paperstrip preloaded with the particles and allowed to migrate along thestrip. Binding of the antigen in the sample to the particles modifiesthe ability of the particles to bind to trapping sites, which containbinding agents for the particles such as antigens or antibodies, furtheralong the strip. Accumulation of the coloured particles at these sitesresults in colour development are dependent on the concentration ofcompeting antigen in the sample. Other dipstick methods may employantibodies covalently bound to paper or membrane strips to trap antigenin the sample. Subsequent reactions employing second antibodies coupledto enzymes such as horse radish peroxidase and incubation withsubstrates to produce colour, fluorescent or chemiluminescent lightoutput will enable quantitation of antigen in the sample.

As discussed in the following examples, radioimmunoassay (RIA) is acurrently preferred laboratory technique. In one RIA a radiolabelledantigen and unlabelled antigen are employed in competitive binding withan antibody. Common radiolabels include ¹²⁵I, ¹³¹I, ³H and ¹⁴C.

Radioimmunoassays involving precipitation of BNP-SP with a specificantibody and radolabelled antibody binding protein can measure theamount of labelled antibody in the precipitate as proportional to theamount of BNP-SP in the sample. Alternatively, a labelled BNP-SP isproduced and an unlabelled antibody binding protein is used. Abiological sample to be tested is then added. The decrease in countsfrom the labelled BNP-SP is proportional to the amount of BNP-SP in thesample.

In RIA it is also feasible to separate bound BNP-SP from free BNP-SP.This may involve precipitating the BNP-SP/antibody complex with a secondantibody. For example, if the BNP-SP antibody complex contains rabbitantibody then donkey anti-rabbit antibody can be used to precipitate thecomplex and the amount of label counted. For example in an LKB,Gammamaster counter. See Hunt et al.²²

The methods of the invention further comprise measuring the levels ofone or more non-BNP-SP markers of the ACD, cardiac transplant rejection,or ACD/pulmonary disorder. The level of the other marker or markers canbe compared to mean control levels from a control population. Adeviation in the measured level from the mean control level ispredictive or diagnostic of ACD or cardiac transplant rejection.

While the methods of the invention have been described with respect to ahigher level or increase in BNP-SP levels being indicative of ACD, orcardiac transplant rejection, it is also possible that in some events ordisorders the levels of BNP-SP will fall. Measuring deviations below acontrol level are also contemplated.

Other markers which are particularly useful herein include troponin T,troponin I, creatin kinase MB, myoglobin, BNP, NT-BNP LDH, aspartateaminotransferase, H-FABP, endothelin, adrenomedullin, rennin andangiotensin II¹. These markers are all implicated in cardiac dysfunctionor disease. Correlating the level of BNP-SP with other markers canincrease the predictive, diagnostic or monitoring value of BNP-SP. Inthe case of ACD, cardiac transplant rejection or ACD/pulmonary disordercombining BNP-SP marker levels with known cardiac markers can increasethe predictive or diagnostic value of a patient outcome.

Analysis of a number of peptide markers can be carried outsimultaneously or separately using a single test sample. Simultaneous,two or multi-site format assays are preferred. Multiplex bead,microassay or biochip systems are particularly useful. The beads, assaysor chips can have a number of discreet, often addressable locations,comprising an antibody to one or more markers including BNP-SP. The oneor more markers include more than one BNP-SP marker. For example, it maybe useful to assay for N-terminal and C-terminal BNP-SP fragments andcombine the assay results. Many other such marker combinations arefeasible. US2005/0064511 provides a description of chips and techniquesuseful in the present invention. Luminex provides a multiplex beadsystem useful in the present invention.

Where a subject is to be monitored, a number of biological samples maybe taken over time. Serial sampling allows changes in marker levels,particular BNP-SP to be measured over time. Sampling can provideinformation on the approximate onset time of an event, the severity ofthe event, which therapeutic regimes may be appropriate, response totherapeutic regimes employed, and long term prognosis. Analysis may becarried out at points of care such as in ambulances, doctors offices, onclinical presentation, during hospital stays, in outpatients, or duringroutine health screening.

The methods of the invention may also be performed in conjunction withan analysis of one or more risk factors such as but not limited to age,weight, sex and family history of events such as cardiac events. Testresults can also be used in conjunction with the methods of theinvention. For example, ECG results and clinical examination. Astatistically significant increase in circulating level of BNP-SP,together with one or more additional risk factors or test results may beused to more accurately diagnose or prognose the subject's condition.

The methods herein can also be used as a guide to therapy. For examplewhat therapies to initiate and when, therapy monitoring, detection ofpositive or adverse effects of therapy, for example heart toxicity ofantimitotic drugs, and adjustment of therapeutic regimes if and whenrequired dependent on results. This can improve short, medium and longterm outcomes for patients. For a guide to treatments see Troughton etal.⁸

Acute Cardiac Disorders

The applicants have shown that concentrations of the full-length BNP-SPmolecule (1-26) and various fragments thereof are correlated with acutecardiac disorders. Moreover, BNP-SP levels are at their highest uponclinical presentation in the case of patients presenting with suspectedacute myocardial infarction (AMI). Patients presenting with acutecardiac disorders, and in particular acute cardiac ischemia may or maynot experience subsequent myocardial infarction (MI). The group whichdoes not experience MI can not be readily diagnosed using currentclinical techniques and markers. For the first time, the applicants havetherefore provided a useful early and specific marker for myocardialdamage associated with MI. This may allow the early diagnosis ofmyocardial damage due to adverse events (AEs) and allow a physician todistinguish such cases from other acute coronary syndromes as well asfrom other causes of a chest pain. For example angina, gastro-intestinaldisease, lung/pleural disorders and the like. This significantlyshortens the window of 6 hours to 12 hours currently experienced waitingfor elevation of levels of current cardiac biomarkers such as myoglobin,CK-MB, TnT and TnI. A more precise diagnosis and treatment can‘therefore be effected’ earlier, reducing morbidity and mortality andgiving better prognostic outcomes.

The invention has particular application in monitoring reperfusiontreatment in cardiac patients. Reperfusion treatment commonly includespercutaneous coronary intervention (eg angioplasty) and/orpharmacological treatment. Thrombolytic drugs for revascularisation arecommonly employed in pharmacological treatment. Adjunctive therapiesinclude anticoagulant and anti-platelet therapies. Reperfusion treatmentis most effective when employed as soon as possible after diagnosis.BNP-SP testing to accelerate diagnosis allows prompt introduction ofreperfusion treatment. Effectiveness of treatment can also be monitoredby repeat testing, and therapy adjusted as appropriate. For acomprehensive discussion of reperfusion treatment see Braunwald et alherein¹.

Cardiac Disease

The methods of the invention can also be used to diagnose or predictcardiac disease in a subject.

The applicants have shown that in patients with acute cardiac disordersthe levels of BNP-SP remain elevated for at least 6 weeks after acardiac event. It is similarly predictable that patients with cardiacdisease or at risk of same will exhibit a higher level of BNP-SP thanmean control levels in a control population. Unlike BNP, the applicantshave shown that levels of BNP-SP are not affected by the age of thepopulation. This suggests BNP-SP has broad applications as a marker ofcardiac disease.

Cardiac Transplant Rejection

The invention also has applications in monitoring heart transplant,commonly a cardiac allograft transplant, rejection through regulartissue biopsy during and after transplant using BNP-SP measurements. Anincrease in BNP-SP levels measured within two hours of heart transplantrelative to a control level may be predictive or diagnostic of arejection episode.

The present invention also provides an assay for BNP-SP in a biologicalsample obtained from a subject within two hours from onset of, or withintwo hours of clinical presentation with ACD, cardiac transplantrejection or ACD/pulmonary disorder, the assay comprising detecting andmeasuring the level of BNP-SP in the sample using any known methods.Preferably, the assay is an in vitro assay. Such methods include all ofthe known assay techniques discussed above as well as gelelectrophoresis techniques, Western blot, gas phase spectroscopy, atomicforce microscopy, surface plasmon resonance, mass spectroscopy but notlimited thereto²³.

In one embodiment the assay comprises one or more nucleic acid sequenceswhich bind to one or more of the BNP-SP nucleic acid sequences of theinvention. A large range of sense and antisense probes and primers canbe designed from the nucleic acid sequences herein. The expression levelof the BNP-SP sequence is identified using known art techniquesdiscussed above. The array can be a solid substrate e.g., a “chip” asdescribed in U.S. Pat. No. 5,744,305 or a nitrocellulose membrane.

Proteins expressed by the BNP-SP marker herein may also be used inassays, and results compared to expression levels of the same proteinexpressed in a normal control sample. Protein presence and quantity maybe assessed using assay formats known in the art and discussed herein.

The presence of BNP-SP is preferably detected in the sample by bindingBNP-SP to a binding agent such as an antibody of the invention andmeasuring the presence of the amount of bound BNP-SP.

As noted above, antibodies selective for BNP-SP including variants andfragments thereof form a further aspect of the invention and theantibodies may be prepared by the techniques discussed above. Theantibodies are useful in the methods and assay of the invention.

In a further aspect, the invention provides a kit for predicting,diagnosing or monitoring acute cardiac disorder (ACD), cardiactransplant rejection, or ACD/pulmonary disorder, comprising a BNP-SPbinding agent of the invention, wherein the kit is for use with abiological sample obtained from a subject within two hours of onset of,or clinical presentation with ACD, cardiac transplant rejection, orACD/pulmonary disorder.

The invention also provides a kit for predicting, diagnosing ormonitoring an acute cardiac disorder (ACD), cardiac transplantrejection, or an ACD/pulmonary disorder comprising a binding agent ofthe invention, wherein the kit is calibrated to measure BNP-SP levels inthe range of 0.1 to 500 pmol/L, preferably 1 to 400 pmol/L, preferably10 to 350 pmol/L, preferably 20 to 300 pmol/L, preferably 25 to 250pmol/L, preferably 30 to 180 pmol/L, preferably 35 to 150 pmol/L,preferably 40 to 120 pmol/L.

Calibration of assays can be effected according to known art techniques,for example using blood samples with known levels of BNP-SP, or a set ofcalibrates with different known levels of BNP-SP in each. Test stripsfor use in diagnostic kits are commonly calibrated during manufacture.See for example U.S. Pat. No. 6,780,645. The kit is useful for measuringthe level of BNP-SP in a biological sample. The detection reagents maybe oligonucleotide sequences complementary to BNP-SP or a fragment ofthe BNP-SP marker, or antibodies which bind to the polypeptides encodedby the marker. The reagents may be bound to a solid matrix as discussedabove or packaged with reagents for binding them to the matrix. Thesolid matrix or substrate may be in the form of beads, plates, tubes,dip sticks, strips or biochips all as discussed above.

Detection reagents include wash reagents and reagents capable ofdetecting bound antibodies (such as labelled secondary antibodies), orreagents capable of reacting with the labelled antibody.

The kit will also conveniently include a control reagent (positiveand/or negative) and/or a means for detecting the nucleic acid orantibody. Instructions for use may also be included with the kit, suchas taking a biological sample from a subject within two hours of onsetor presentation with ACD, cardiac transplant rejection or ACD/pulmonarydisorder, measuring the level of BNP-SP in the sample, comparing same toa control level and associating the result with cardiac status.Generally an increase in the BNP-SP marker level from a control isindicative of ACD or cardiac transplant rejection, or ACD as opposed toa pulmonary disorder.

Most usually, the kits will be formatted for assays known in the art,and more usually for PCR, Northern hybridization or Southern ELISAassays, as are known in the art.

The kits may also include one or more additional markers for ACD,transplant rejection, or ACD/pulmonary disorders. In the case of ACS theadditional marker may include one or more of troponin T, troponin I,creatin kinase MB, myoglobin, BNP, NT-BNP, LDH, aspartateaminotransferase, H-FABP, endothelin, adrenomedullin, rennin andongrotensin II. In one embodiment all of the markers are included in thekit.

The kit will be comprised of one or more containers and may also includecollection equipment, for example, bottles, bags (such as intravenousfluids bags), vials, syringes, and test tubes. At least one containerholds a product which is effective for predicting, diagnosing, ormonitoring ACD (particularly ACS), transplant rejection, orACD/pulmonary disorder. The product is usually a nucleic acid molecule,polypeptide or a binding agent of the invention, or a compositioncomprising any of these. In a preferred embodiment, an instruction orlabel on, or associated with, the container indicates that thecomposition is used for predicting, diagnosing, or monitoring ACD(particularly ACS), transplant rejection, or ACD/pulmonary disorders.Other components may include needles, diluents and buffers. Usefully,the kit may include at least one container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution.

Binding agents that selectively bind BNP-SP are desirably included inthe kit. Preferably, the binding agent is an antibody. The antibody usedin the assays and kits may be monoclonal or polyclonal and may beprepared in any mammal as discussed above. The antibodies are preferablyprepared against a native peptide encoded or indicated by a BNP-SPnucleic acid sequence of the invention, BNP-SP (1-26), or a syntheticpeptide based on same, or may be raised against an exogenous sequencefused to a nucleic acid sequence encoding a BNP-SP peptide of theinvention.

In one kit embodiment a BNP-SP detection reagent is immobilized on asolid matrix such as a porous strip to form at least one BNP-SPdetection site. The measurement or detection region of the porous stripmay include a plurality of detection sites, such detection sitescontaining a BNP-SP detection reagent. The sites may be arranged in abar, cross or dot or other arrangement. A test strip may also containsites for negative and/or positive controls. The control sites mayalternatively be on a different strip. The different detection sites maycontain different amounts of immobilized nucleic acids or antibodies eg,a higher amount in the first detection site and lower amounts insubsequent sites. Upon the addition of a test biological sample thenumber of sites displaying a detectable signal provides a quantitativeindication of the amount of BNP-SP present in the sample.

Also included in the kit may be a device for sample analysis comprisinga disposable testing cartridge with appropriate components (markers,antibodies and reagents) to carry out sample testing. The device willconveniently include a testing zone and test result window.Immunochromatographic cartridges are examples of such devices. See forexample U.S. Pat. No. 6,399,398; U.S. Pat. No. 6,235,241 and U.S. Pat.No. 5,504,013.

Alternatively, the device may be an electronic device which allowsinput, storage and evaluation of levels of the measured marker againstcontrol levels and other marker levels. US 2006/0234315 providesexamples of such devices.

In this specification where reference has been made to patentspecifications, other external documents, or other sources ofinformation, this is generally for the purpose of providing a contextfor discussing the features of the invention. Unless specifically statedotherwise, reference to such external documents is not to be construedas an admission that such documents; or such sources of information, inany jurisdiction, are prior art, or form part of the common generalknowledge in the art.

The invention will now be illustrated in a non-limiting way be referenceto the following examples.

Example 1 Methods

All human protocols were approved by the Upper South Regional EthicsCommittee of the Ministry of Health, New Zealand and were performed inaccord with the Declaration of Helsinki.

Chemicals

Synthetic human BNP signal peptides BNP-SP (1-10), BNP-SP(17-26) andBNP-SP(1-26) (SEQ ID NO:1) were synthesised by Mimotopes (Australia).All buffer reagents were purchased from BDH and/or Sigma. BNP-SP (17-26)was synthesised with the C-terminal extended with cysteine fordirectional carrier coupling. BNP-SP (17-26) was also C-terminallyextended with a tyrosyl residue for tracer preparation on the samepeptide.

Human Studies

For the healthy volunteer reference range study, blood samples wereobtained from 13 healthy volunteers (6 woman, average age 43±12 years(range 22-60 years), BMF 24.4±3.9 kg/m²) after an overnight fast.

For analysis of BNP-SP concentrations in acute cardiac injury, westudied 10 consecutive patients (4 woman, average age 70±8 years (range59-79 years)), presenting to the Coronary Care Unit at ChristchurchHospital within 6 h of the onset of chest pain and clear evidence ofST-elevation acute MI, together with a rise then fall in plasma troponinT (TnT). Patients with cardiogenic shock were excluded. Five patientshad previously documented hyperlipidaemia, four had hypertension, onehad an earlier MI, one was being treated for cardiac failure and two haddiabetes mellitus. Medications on admission were diuretics (twopatients), angiotensin-converting enzyme inhibitors (two patients),aspirin (seven patients), β-blockers (two patients). One patient hadprimary percutaneous transluminal coronary angioplasty (PTCA foranterior MI), nine patients received thrombolysis. Seven patients had anECG during the hospital stay. Across all patients, the average ejectionfraction was 54% (range, 24-75%). Average hospital stay was 6.6 days(range, 3-15 days). The time between the onset of chest pain and drawingof the baseline (time 0) venous sample was 3.9±0.3 h. An 18-gaugeintravenous cannula was inserted into a forearm vein for blood sampling.Venous samples (10 ml) were drawn on admission to the Coronary Care Unit(time 0) and thereafter at 0.5, 1, 4, 8, 12, 24 and 72 h as in-patients,and at 1, 6 and 12 weeks as out-patients. Samples were taken into tubeson ice and centrifuged at +4° C. at 2700 g for 5 min and the plasmastored at −80° C. until analysed.

Plasma Extraction

All plasma samples were extracted on SepPak manufacturer waters, USAcartridges as previously described²², dried and stored at −20° C. priorto RIA and HPLC.

Hormone Concentration Analysis

Plasma samples were assayed for TnT, CK-MB and myoglobin usingheterogeneous immunoassays on an Elecsys 2010 using ruthenium-labelledbiotinylated antibodies according to standard manufacturers' protocols,Roche Diagnostics.¹⁸ Immunoreactive (IR) BNP and N-BNP concentrationswere measured using our previously described assays.⁶⁻⁸ BNP-SP wasmeasured by specific RIA as follows:

BNP-SP RIA

For the measurement of putative human BNP-SP IR peptides, we generated anovel and specific RIA directed against amino acids 17-26 of the humanpreproBNP(1-26) signal sequence (SEQ ID NO:1)

Antibody Generation

preproBNPCys²⁵ (17-26) was coupled to malemide treatedN-e-maleimidocaproyloxy succinimide ester (EMCS) derivatised BSA in PBS(pH 7.0) by gentle mixing at room temperature. Coupled peptide wasemulsified with Freund's adjuvant and injected subcutaneously in 2 NewZealand white rabbits over 4-5 sites at monthly intervals. Rabbits werebled 12 days after injection to assess antibody titres until adequatelevels were achieved. For RIA, BNP-SP IR was determined using antiserumat a final dilution of 1:6,000. This antiserum has no detectable crossreactivity with human proBNP(1-13), proBNP(1-76), proANP(1-30), ANP,BNP, endothelin 1, Angiotensin II, Angiotensin(1-7), urotensin. II, CNP,proCNP(1-15), adrenomedullin, urocortin I and urocortin II (all <0.01%).

Iodination and Assay Method

preproBNP Tyr²⁵ (17-26) was iodinated via the Chloramine T method andpurified on reverse phase HPLC as previously described²² All samples,standards, radioactive traces and antiserum solutions were diluted inpotassium based assay buffer.²² The assay incubate consisted of 100 μLsample or standard (0-640 pmol human preproBNP(17-26) combined with 100μL antiserum which was vortexed and incubated at 4° C. for 24 hours.1004 of trace (4000-5000 cpm) was then added and further incubated for24 hours at 4° C. Free and bound immunoreactivities were finallyseparated by solid phase second antibody method (donkey anti-rabbitSac-Cel) and counted in a Gammamaster counter (LKB, Uppsala, Sweden).

High Performance Liquid Chromatography (HPLC)

Plasma extracts were subjected to size-exclusion HPLC (SE-HPLC) at roomtemperature on a TSK-Gel G2000SW peptide column (Toyosoda, Tokyo, Japan)using isocratic conditions of 60% acetonitrile/0.1% trifluoroacetic acid(TFA) at a flow rate of 0.25/ml/minute. Fractions were collected at 1minute intervals and subjected to BNP-SP RIA. The SE-HPLC column wascalibrated using dextran blue (Vo), cytochrome C (˜Mr 12,400), rat BNP45(˜Mr 5,000), angiotensin II (˜Mr 1,045) and glycine (Vt). BNP-SP IRidentified by SE-HPLC/RIA were then further characterised on a BrownleeC₁₈ reverse phase HPLC(RP-HPLC) column (Applied Biosystems, CA) with alinear eluting gradient from 12%-48% acetonitrile/0.1% TFA over 40minutes, at a flow rate 1 ml/minute. One minute fractions werecollected, dried under an air stream and subjected to specific RIA asfor SE-HPLC. RP-HPLC was calibrated using synthetic preproBNP(17-26).

Statistical Analysis

All results are presented as mean±SEM. Time-course data were analysedusing two-way ANOVA for repeated measurements followed by leastsignificant difference post-hoc testing. Correlation analysis of plasmahormone concentrations was carried out using a general linear regressionmodel. In all analyses, a P-value <0.05 was considered significant.

Results

To determine if the 26 amino acid SP of BNP, or fragments derived fromit, are present in circulation of humans, we developed a specificradioimmunoassay (RIA) directed against residues 17-26 ofpreproBNP(1-26) (BNP-SP, FIG. 2). Dilution of plasma extractsdemonstrate parallelism with the standard curve (FIG. 3) and plasmaconcentrations of BNP-SP in healthy humans were 9.6±2.2 pmol/L (n=13).In healthy humans, concentrations of BNP-SP IR in blood do not show asignificant correlation with age (FIG. 4). However, while plasma BNP-SPlevels are similar to those of its sibling peptides BNP and N-BNP, theydo not correlate with either peptide (FIG. 5).

Biochemical analysis of IR plasma BNP-SP by reverse phase (RP) and sizeexclusion (SE) high performance liquid chromatography (HPLC) suggestthat our specific RIA detects fragment(s) of BNP-SP that elute with anapproximate Mr 1,000-2,000 on SE-HPLC and close to the elution time ofsynthetic BNP-SP on RP-HPLC (FIG. 6).

Having established that IR BNP-SP peptides are present in human plasmawe then measured serial concentrations of IR BNP-SP in patients withdocumented AMI (n=10, FIG. 7). Highest concentrations of IR BNP-SP wereobserved at hospital admission and slowly dropped to stable levels over6 weeks. Importantly, average peak levels at admission were 7-foldhigher (range 4-12) than levels in normal healthy volunteers andremained 3-fold higher up to 6 weeks. This pattern stands in contrast tothat of BNP and N-BNP whose peaks levels do not occur until 24 hourspost-admission (FIG. 7). Peak concentrations of myoglobin occurred 1-2hours after hospital admission, whereas peak TnT and CK-MB levels werenot attained until 8-12 hours after admission.

Example 2

Thirty two patients with clinically stable suspected ACS werecatheterized and blood samples from multiple organ sites: these were thefemoral artery (FA), hepatic vein (HV, inferior vena cava (IVC), cardiaccoronary sinus vein (CS) and pulmonary artery (PA). Blood was collectedinto chilled EDTA tubes, prepared from plasma by centrifugation and theplasma submitted to BNP-SP RIA. FIG. 9 clearly shows that the highestsite of BNP-SP concentration is the CS, the vein draining the heart,especially the ventricles. This is strong evidence that the heart is thepredominant site of BNP-SP secretion and is consistent with the knowngene expression pattern of BNP, being highest in the heart.

CONCLUSION

Circulating BNP-SP concentrations in clinically stable patients arederived from cardiac sources. The significant cardiac secretion, isconsistent with BNP-SP being a cardiac hormone.

DISCUSSION

This evidence is the first to document the signal peptide of preproBNPas being present in the circulation and extracellular space within twohours of a patient presenting with ACD or within two hours of the onsetof ACD. We show in the first instance that the measurement of BNP-SP inblood has potential as a rapid biomarker of acute cardiac ischemiaand/or subsequent injury and in the second instance, that measurement ofBNP-SP after the event has potential merit as a marker of long termprognosis and outcome.

Those skilled in the art will of course appreciate that the abovedescription is provided by way of example and that the invention is notlimited thereto.

REFERENCES

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All references and citations in this list and throughout thespecification including patent specifications are hereby incorporated intheir entirety.

We claim:
 1. An assay method for diagnosing an acute cardiac disorder ina subject, the method comprising: a. obtaining a biological sample fromthe subject; b. contacting the sample with a BNP-SP fragment bindingagent; c. determining the level of a BNP-SP fragment in the biologicalsample obtained from the subject; and d. comparing the level of saidBNP-SP fragment in the sample with the level of said BNP-SP fragment ina control or reference value, wherein a level of BNP-SP fragment in thesample that is higher than the control level is diagnostic of an acutecardiac disorder.
 2. A method according to claim 1, wherein said methodis used to evaluate or monitor a response to treatment of an acutecardiac disorder, wherein said assay method is carried out on one ormore biological samples from said subject and a change in the measuredlevel of BNP-SP fragment from said control or reference value isdetermined, said method further comprising the step of determiningwhether to adjust treatment for said acute cardiac disorder based on achange in the measured level of BNP-SP fragment from the control orreference value and, optionally, adjusting said treatment.
 3. A methodaccording to claim 1, which further comprises the step of providingtherapy to the subject for treatment of an acute cardiac disorder.
 4. Amethod according to claim 1 that further comprises the step ofevaluating one more risk factors and/or test results in conjunction withdetermining the level of BNP-SP fragment in the sample.
 5. A methodaccording to claim 1, wherein the control is an individual or group fromwhich BNP-SP fragment samples are obtained and a mean BNP-SP fragmentlevel is determined.
 6. A method according to claim 5, wherein theindividual or group comprises normal healthy individuals or a group ofindividuals not known to be suffering from an acute cardiac disorder. 7.A method according to claim 5, wherein the control BNP-SP fragmentlevels are between 0 to 15 pmol/L.
 8. A method according to claim 5,wherein the control or reference value is derived from BNP-SP fragmentlevel in a cardiac control population.
 9. A method according to claim 8,wherein the BNP-SP fragment level in the cardiac control population isin the order of 1.5 to 3, 2 to 3, or 2.5 to 3 times higher than BNP-SPfragment levels in a normal control population.
 10. A method accordingto claim 1, wherein repeat BNP-SP fragment level determinations arecarried out on separate samples from the subject.
 11. A method accordingto claim 1 wherein a sample level of BNP-SP fragment in the range of anyof 20 to 300 pmol/L, 25 to 250 pmol/L, 20 to 180 pmol/L, 30 to 180pmol/L, 35 to 150 pmol/L, 40 to 130 pmol/L, 40 to 120 pmol/L, 40 to 90pmol/L, 45 to 80 pmol/L, 45 to 160 pmol/L, 50 to 100 pmol/L and 50 to200 pmol/L is diagnostic of an acute cardiac disorder.
 12. A methodaccording to claim 1, wherein a level of BNP-SP fragment in the samplewhich is five to eight times higher, or four to ten times higher, thanthe control or reference level is diagnostic of acute cardiac disorder.13. A method according to claim 1, wherein the acute cardiac disorder isunstable angina.
 14. A method according to claim 1, wherein the acutecardiac disorder is an acute myocardial infarction.
 15. A methodaccording to claim 14, wherein the acute myocardial infarction is anacute myocardial infarction with ST-elevation on presenting ECG.
 16. Amethod according to claim 14, wherein the acute cardiac disorder isnon-ST elevated myocardial infarction.
 17. A method as claimed in claim1 wherein the acute cardiac disorder is acute cardiac ischemia.
 18. Amethod according to claim 1, wherein the acute cardiac disorder is anacute cardiac injury, acute cardiac damage resulting from acute drugtoxicity, an acute cardiomyopathy or a cardiac transplant rejectionepisode.
 19. A method according to claim 1, wherein the biologicalsample is a blood.
 20. A method according to claim 1, wherein thebiological sample is plasma.
 21. A method according to claim 1, whereinthe level of the BNP-SP fragment in the biological sample is determinedby immunoassay.
 22. A method according to claim 21, wherein theimmunoassay is a competitive binding assay, a non-competitive assay, asandwich assay, a fluoroimmunoassay, a immunofluorometric assay, animmunoradiometric assay, a luminescence assay or a chemiluminescenceassay.
 23. A method according to claim 21, wherein the immunoassay is aquantitative immunoassay.
 24. A method according to claim 21, whereinthe immunoassay comprises an antibody or an antibody binding fragment.25. A method according to claim 24, wherein the antibody or antibodybinding fragment is attached to a solid phase.
 26. A method according toclaim 21, wherein the immunoassay comprises a labeled antibody or alabeled antibody binding fragment.
 27. A method according to claim 1,wherein the BNP-SP fragment level is evaluated by mass spectroscopy. 28.A method according to claim 1, wherein the BNP-SP fragment is BNP-SP(17-26) (SEQ ID NO:19).
 29. A method according to claim 1, wherein theBNP-SP fragment is selected from the group consisting of BNP-SP (1-10)(SEQ ID NO:13), BNP-SP (1-17) (SEQ ID NO:15) and BNP-SP (12-23) (SEQ IDNO:17).
 30. A method according to claim 1, wherein the assay method iscarried out using a device for sample analysis comprising a disposabletesting cartridge.
 31. A method according to claim 1, which furthercomprises the step of determining the level of one or more non-BNP-SPmarkers associated with an acute cardiac disorder.
 32. A methodaccording to claim 31 wherein the non-BNP-SP marker is selected from thegroup consisting of troponin, troponin T, troponin I, creatinekinase-MB, myoglobin, BNP, NT-BNP, and H-FABP.